Angiogenesis inhibitor

ABSTRACT

An angiogenesis inhibitor comprising a cysteine protease inhibitory compound. As the preferable cysteine protease inhibitory compound, epoxysuccinic acid compounds, peptide halohydrazide compounds, calpain inhibitory compounds, compounds of the formula (I)                    
     and compounds of the formula (VI)                    
     can be used. The angiogenesis inhibitor of the present invention suppresses new formation of blood vessels in the living tissues, so that it can be used as a superior therapeutic or prophylactic agent of angiogenesis associated with wound healing, inflammation, growth of tumor and the like; and angiogenesis as seen in diabetic retinopathy, prematurity retinopathy, retinal venous occlusion, senile discoid macular degeneration and the like, as well as for prevention of metastasis of tumors.

This application is a divisional of Ser. No. 09/243,822 filed Feb. 3,1999, which was a divisional of Ser. No. 08/740,069 filed Oct. 24, 1996,now U.S. Pat. No. 6,057,290.

TECHNICAL FIELD

The present invention relates to an angiogenesis inhibitor comprising acysteine protease inhibitory compound.

BACKGROUND OF THE INVENTION

An angiogenesis is a phenomenon wherein new blood vessels are created toform a new vascular network in the living body. The angiogenesis isfound under normal physiological environment such as genesis andreproduction with regard to embryo, fetus, placenta, uterus and thelike. It is also a pathologic phenomenon which accompanies woundhealing, inflammation, growth of tumor and the like, and which isophthalmologically seen in diabetic retinopathy, prematurityretinopathy, retinal venous occlusion, senile discoid maculardegeneration and the like.

The angiogenesis greatly varies depending on the function and growth ofendothelial cells, and is considered to be a cascade reaction whichproceeds in the smallest vein along the following steps. That is, newblood vessels are presumably formed as a result of consecutiveelementary reactions of (1) activation of vascular endothelial cellswhich are in the stage of rest upon differentiation, (2) destruction ofcell matrix such as basement membrane by endothelial cells whichexpressed protease activity, (3) migration of endothelial cells, (4)proliferation of endothelial cells and (5) tube-formation bydifferentiation of endothelial cells [T. Oikawa, Drug News Perspest,Vol. 6, pp. 157-162 (1993)]. Each step of these reactions has beenclarified to be promoted by angiogenesis promoters. Such angiogenesispromoters include, for example, blood vessel inducing factors [e.g.,tumor angiogenetic factor (TAF)] secreted from tumor tissues, and growthfactors such as fibroblast growth factor (FGF) present in various normaltissues, endothelial cell growth facor derived from platelets andvascular endothelial cell growth factor. In addition, cytokine,prostaglandine, monobutylin and angiogenine reportedly have similardirect or indirect effects [M. Klagsbrun et al., Annu. Rev. Physiol.,Vol. 53, pp. 217-239 (1991)].

A substance which suppresses such angiogenesis include angiostaticsteroids [Folkman, J. et al., Science, Vol. 221, p. 719 (1983)] such ascortisone which inhibits growth of endothelial cells;medroxyproge-sterone acetate which inhibits production of plasminogenactivator by endothelial cells; fumagillin acid derivatives whichinhibit proliferation of endothelial cells and tube-formation;polysaccharide sulfate SD-4152 which inhibits proliferation andmigration of endothelial cells; and retinoic acid which is responsiblefor modification of endothelial cell differentiation [Tsutomu Oikawa,Kekkan to Naihi, vol. 2, pp. 470-480 (1992)].

However, the above-mentioned drugs which inhibit angiogenesis have notbeen complete therapeutic agents for clinically suppressingangiogenesis, since some of them cause strong side-effects, therebyposing problems in terms of safety, and others only show insufficienteffects.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide apharmaceutical agent which provides strong angiogenesis-inhibitoryeffects.

According to the present invention, there has been provided (1) anangiogenesis inhibitor comprising a cysteine protease inhibitorycompound.

Cysteine protease is a protease having a cysteine residue in the activesite of the enzyme molecule and includes such species as cathepsin B, H,and L and dipeptidyl peptidase, all of which are lysosomal enzymefractions, and calpain which occurs in the cytoplasm, among others.Though much remains to be explored about the physiological roles ofthese enzymes, a considerable amount of light has been cast on theirroles in recent years. For example, calpain is known to be a proteaseubiquitous in life, which is activated by calcium ions and has theoptimum pH in neutral. As elucidated to this day, it takes part indegradation of the skeletal protein of cells, activation of inert cellprecursors such as protein kinase C, and degradation of receptorproteins. It has also been shown that the abnormality of this enzymeactivity is involved in many diseases. For example, its involvement inrefractory diseases such as cerebral apoplexy (stroke), subarachnoidhemorrhage, Alzheimer's disease, ischemic diseases, muscular dystrophy,cataract, platelet aggregation disorder, arthritis, and osteoporosis,among other diseases. [Trends in Pharmacological Science, Vol. 15, p.412 (1994)].

The angiogenesis inhibitor of the present invention may have thefollowing modes.

(2) The angiogenesis inhibitor of above (1) wherein the cysteineprotease inhibitory compound is a calpain inhibitory compound.

(3) The angiogenesis inhibitor of above (2) wherein the calpaininhibitory compound is at least one compound selected from calpastatinand calpastatin peptide.

(4) The angiogenesis inhibitor of above (3) wherein the calpastatinpeptide is at least one compound selected from peptides having an aminoacid sequence of the following formula:

-Gly-A-Tyr-Arg-

wherein A is -Lys-Arg-Glu-Val-Thr-Ile-Pro-Pro-Lys-(SEQ ID NO:1),-Lys-Arg-Glu-Val-Thr-Leu-Pro-Pro-Lys-(SEQ ID NO:2),-Glu-Asp-Asp-Glu-Thr-Ile-Pro-Ser-Glu-(SEQ ID NO:3),-Glu-Asp-Asp-Glu-Thr-Val-Pro-Pro-Glu-(SEQ ID NO:4),-Glu-Asp-Asp-Glu-Thr-Val-Pro-Ala-Glu-(SEQ ID NO:5),-Glu-Lys-Glu-Clu-Thr-Ile-Pro-Pro-Asp- or-Glu-Arg-Asp-Asp-Thr-Ile-Pro-Pro-Glu-(SEQ ID NO:7).

(5) The angiogenesis inhibitor of above (4) wherein the calpastatinpeptide has an amino acid sequence of the following formula:

Asp-Pro-Met-Ser-Ser-Thr-Tyr-Ile-Glu-Glu-Leu-Cly-Lys-Arg-Glu-Val-Thr-Ile-Pro-Pro-Lys-Tyr-Arg-Glu-Leu-Leu-Ala(SEQ ID NO:8).

(6) The angiogenesis inhibitor of above (2) wherein the calpaininhibitory compound inhibits Ca²⁺-binding site having a high homologywith calmodulin in calpain.

(7) The angiogenesis inhibitor of above (6) wherein the compound whichinhibits Ca²⁺-binding site having a high homology with calmodulin is atleast one compound selected from calmodulin antagonistic compounds.

(8) The angiogenesis inhibitor of above (1) wherein the cysteineprotease inhibitory compound is at least one compound selected from thegroup consisting of epoxysuccinic peptide compounds, peptide aldehydecompounds, peptide halomethane compounds, peptide diazomethanecompounds, peptide halohydrazide compounds, peptide disulfide compounds,peptide ketoamide compounds and isocoumarine compounds.

(9) The angiogenesis inhibitor of above (8) wherein the cysteineprotease inhibitory compound is an epoxysuccinic peptide compound.

(10) The angiogenesis inhibitor of above (9) wherein the cysteineprotease inhibitory compound is an epoxysuccinic peptide compound of theformula (I):

wherein

R¹ is an optionally esterified carboxy or an optionally substitutedcarboxamide;

R² is a hydrogen or a lower (unless otherwise specified, “lower” means“having 1 to 6 carbon atoms” in the present specification) alkyl orforms a ring together with R3 or R⁴;

R³ and R⁴ are the same or different and each is a hydrogen, anoptionally substituted lower alkyl, an optionally substituted sulfide,or R³ and R⁴ combindly form a ring;

R⁵ is a substituted phenyl of the formula (II)

wherein R⁶ is halogen atom or alkoxy, or a substituted sulfonyl of theformula (III)

S O₂-R⁷  (III)

wherein R⁷ is aryl optionally substituted by lower alkyl or optionallysubstituted amino; and

n is 0 or 1, or a salt thereof.

(11) The angiogenesis inhibitor of above (10) wherein R¹ is anoptionally esterified carboxy, or carboxamide optionally substituted byhydroxy or aralkyloxy.

(12) The angiogenesis inhibitor of above (10) wherein R² is hydrogen ormethyl.

(13) The angiogenesis inhibitor of above (10) wherein R² and R³, or R²and R⁴ combinedly form a pyrrolidine ring.

(14) The angiogenesis inhibitor of above (10) wherein R³ and R⁴ are thesame or different and each is hydrogen, lower alkyl optionallysubstituted by aromatic group or carbamoyl, or sulfide optionallysubstituted by acylamino.

(15) The angiogenesis inhibitor of above (10) wherein R³ and R⁴combinedly form a cyclopentane ring.

(16) The angiogenesis inhibitor of above (10) wherein R⁶ of the formula(II) is chlorine or fluorine.

(17) The angiogenesis inhibitor of above (10) wherein R⁷ of the formula(III) is phenyl or dimethylamino optionally substituted by lower alkyl.

(18) The angiogenesis inhibitor of above (8) wherein the cysteineprotease inhibitory compound is a peptide aldehyde compound.

(19) The angiogenesis inhibitor of above (18) wherein the peptidealdehyde compound is leupeptin.

(20) The angiogenesis inhibitor of above (18) wherein the peptidealdehyde compound is a compound of the formula (VI):

wherein R¹¹ is an optionally substituted aryl having 6 to 10 carbonatoms; R¹² and R¹³ are the same or different and each is a hydrogen, aC₁-C₄ alkyl, or R¹² and R¹³ combinedly form a ring having 3 to 7 carbonatoms; and R¹⁴ is a lower alkyl optionally substituted by aryl,cycloalkyl or aromatic heterocycle, or a salt thereof.

(21) The angiogenesis inhibitor of above (20) wherein R¹¹ is phenyl ornaphthyl optionally substituted by fluorine, chlorine or methyl.

(22) The angiogenesis inhibitor of above (21) wherein R¹¹ is a memberselected from ⁴-fluorophenyl, 4-chlorophenyl, p-tolyl and 2-naphthyl.

(23) The angiogenesis inhibitor of above (20) wherein R¹² is propyl,isopropyl or tert-butyl, and R¹³ is hydrogen.

(24) The angiogenesis inhibitor of above (23) wherein R¹² is isopropyland R¹³ is hydrogen.

(25) The angiogenesis inhibitor of above (20) wherein R¹² and R¹³combinedly form cyclohexylidene.

(26) The angiogenesis inhibitor of above (20) wherein R¹⁴ is isobutyl,benzyl, cyclohexylmethyl or indol-3-ylmethyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A), FIG. 1(B) and FIG. 1(C) show cornea of guinea pig observedwith a slit lamp, at 9 days after implantation of bFGF-containingpellet, and 27 mer calpastatin peptide (0.03 μmole and 0.1μmole)-containing pellet together with bFGF-containing pellet.

FIG. 2(A) and FIG. 2(B) show cornea of guinea pig observed with a slitlamp, at 9 days after implantation of bFGF-containing pellet, andleupeptin (0.1 μmole)-containing pellet together with bFGF-containingpellet.

FIG. 3 is a graph showing the wet weight of cornea of guinea pigobtained at 9 days after implantation of bFGF-containing pellet, and 27mer calpastatin peptide (0.03 μmole and 0.1 μmole)-containing pellet orleupeptin (0.1 μmole)-containing pellet together with bFGF-containingpellet.

FIG. 4 is a graph showing an amount of albumin in cornea of guinea pigobtained at 9 days after implantation of bFGF-containing pellet, and 27mer calpastatin peptide (0.03 μmole and 0.1 μmole)-containing pellet orleupeptin (0.1 μmole)-containing pellet together with bFGF-containingpellet.

DISCLOSURE OF THE INVENTION

The cysteine protease inhibitor to be used for the angiogenesisinhibitor of the present invention may be any compound as long as it caninhibit cysteine protease. Examples of such compound includeepoxysuccinic peptide compounds such as(+)-(2S,3S)-3-[[[1-[[[4-[(aminoiminomethyl)amino]butyl]amino]carbonyl]-3-methylbutyl]amino]-carbonyl]-2-oxyranecarboxylicacid (E-64),(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butylcarbamoyl]-2-oxyranecarboxylicacid (E-64c) and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)-butylcarbamoyl]-2-oxyranecarboxylate(E-64d); peptide aldehyde compounds such as leupeptin, calpeptin,Ac-Leu-Leu-nLeu-H (calpain inhibitor peptide I), Ac-Leu-Leu-nMet-H(calpain inhibitor peptide II), Z-Val-Phe-H (MDL28170) andBoc-Leu-Nle-H; peptide halomethane compounds such as Z-Leu-Leu-Tyr-CH₂F;peptide diazomethane compounds such as Z-Leu-Leu-Tyr-CHN₂; peptidehalohydrazide compounds such as Z-3-I-Tyr-NHNHCOCH₂I; peptide disulfidecompounds such as Leu-Leu-(3-nitro-2-pyridinesulfinyl)Cys-NH₂; peptideketoamide compounds such as Z-Leu-Abu-CONHEt (AK275); and isocoumarinecompounds such as7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarine.

The cysteine protease inhibitory compound to be used for theangiogenesis inhibitor of the present invention may be a substance whichspecifically inhibits calpain or cathepsin B, H and L, from amongcysteine proteases. Examples of the substance which specificallyinhibits calpain include calpastatin, calpastatin peptide and the like.

The above-mentioned calpastatin peptide is preferably a peptide havingan amino acid sequence of the following formula:

-Gly-A-Tyr-Arg-

wherein A is -Lys-Arg-Glu-Val-Thr-Ile-Pro-Pro-Lys-,-Lys-Arg-Glu-Val-Thr-Leu-Pro-Pro-Lys-,-Glu-Asp-Asp-Glu-Thr-Ile-Pro-Ser-Glu-,-Glu-Asp-Asp-Glu-Thr-Val-Pro-Pro-Glu-,-Glu-Asp-Asp-Glu-Thr-Val-Pro-Ala-Glu-,-Glu-Lys-Glu-Glu-Thr-Ile-Pro-Pro-Asp- or-Glu-Arg-Asp-Asp-Thr-Ile-Pro-Pro-Glu-, particularly, a peptide (27 mercalpastatin peptide) having an amino acid sequence of the followingformula:Asp-Pro-Met-Ser-Ser-Thr-Tyr-Ile-Glu-Glu-Leu-Gly-Lys-Arg-Glu-Val-Thr-Ile-Pro-Pro-Lys-Tyr-Arg-Glu-Leu-Leu-Ala.

The cysteine protease inhibitory compound to be used for theangiogenesis inhibitor of the present invention may contain a substancewhich inhibits Ca²⁺-binding site having a high homology with calmodulin.Examples of such substance include calmodulin antagonistic compoundssuch as melitin, calmidazolium, trifluoroperazine andN-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7),and ethylenediaminetetraacetic acid (EDTA).

These cysteine protease inhibitory compounds may be used alone or incombination.

As an epoxysuccinine peptide compound having strong cysteine proteaseinhibitory effect, a new compound of the following formula may be used.

wherein each symbol is as defined above.

Referring to the above formula (I), the optionally esterified carboxyrepresented by R¹ includes but is not limited to carboxy andalkoxycarboxy. The alkoxy moiety of said alkoxycarboxy may be C₁-C₆alkoxy and preferably C₁-C₄ alkoxy, such as methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy, sec-butoxy, and tert-butoxy. Particularlypreferred is ethoxy.

The substituent for said optionally substituted carboxamide representedby R¹ includes hydroxy, alkoxy (methoxy, ethoxy, propoxy, etc.), andaralkyloxy (benzyloxy etc.). Preferred are hydroxy and benzyloxy.

The lower alkyl for R² includes C₁-C₆ linear- or branched alkyl orpreferably C₁-C₄ alkyl, such as methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, tert-pentyl, n-hexyl, isohexyl, 4-methylpentyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and2-ethylbutyl. Preferred are hydrogen and methyl.

The ring that may be formed combinedly by R² and either R³ or R⁴includes but is not limited to aziridine, azetidine, pyrrolidine, andpiperidine. Particularly preferred is pyrrolidine.

The alkyl of said optionally substituted lower alkyl for R³ and R⁴includes C₁-C₆ linear or branched alkyl such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,isopentyl, neopentyl, tert-pentyl, n-hexyl, 4-methylpentyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and2-ethylbutyl. Preferred are methyl, ethyl, isobutyl, and sec-butyl. Thesubstituent optionally present on said alkyl includes an aromatic ringand carbamoyl. The aromatic ring includes aromatic carbocycles such asbenzene ring and aromatic heterocycles such as indole ring. Particularlypreferred is benzene ring.

The sulfide group of said optionally substituted sulfide for R³ and R⁴includes alkylthioalkyl and preferably C₁-C₄ alkyl-thio-C₁-C₄ alkyl,such as dimethyl sulfide, diethyl sulfide, dipropyl sulfide, dibutylsulfide, dipentyl sulfide, dihexyl sulfide, methylethyl sulfide,methylpropyl sulfide, and ethylbutyl sulfide. Preferred are dimethylsulfide and methylethyl sulfide. The substituent optionally present onsaid sulfide includes acylamino. The acylamino includes but is notlimited to formylamino, acetylamino, propionylamino, butyrylamino,isobutyrylamino, valerylamino, isovalerylamino, pivaloylamino, andn-hexanoylamino. Preferred is acetylamino.

The ring optionally formed combinedly by R³ and R⁴ includescyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, etc.Particularly preferred is cyclopentane.

Referring to the substituent R⁶ for said substituted phenyl of formula(II), the halogen includes but is not limited to fluorine, chlorine,bromine, and iodine. Preferred are fluorine and chlorine. The halogenmay be bonded at any of meta, para, and ortho positions.

Referring further to the substituent R⁶ for said substituted phenyl offormula (II), the alkoxy includes C₁-C₆ alkoxy and preferably C₁-C⁴alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,sec-butoxy, and tert-butoxy. Particularly preferred is methoxy.

Referring to the substituent R⁷ for the substituted sulfonyl of formula(III), the aryl of said aryl optionally substituted by lower alkylincludes but is not limited to phenyl and naphthyl. The lower alkyloptionally substituting said aryl includes methyl, ethyl, propyl,isopropyl, butyl, etc. and may be bonded at any position of the arylgroup.

Referring further to the substituent R⁷ for said substituted sulfonyl offormula (III), the amino includes amino and amino substituted by one ortwo C₁-C₆ linear, branched or cyclic alkyl, such as methylamino,dimethylamino, ethylamino, diethylamino, propylamino, dipropylamino,isopropylamino, diisopropylamino, butylamino, dibutylamino andcyclohexylamino. Particularly preferred is dimethylamino.

In the context of the present invention, the salt of the compound offormula (I) is preferably a physiologically acceptable salt, thusincluding salts with inorganic bases, salts with organic bases, saltswith inorganic acids, salts with organic acids, and salts with basic oracidic amino acids. The preferred inorganic base salt includes alkalimetal salts such as sodium salt and potassium salt, alkaline earth metalsalts such as calcium salt and magnesium salt, aluminum salt, andammonium salt. The preferred organic base salt includes salts withtrimethylamine, pyridine, picoline, ethanolamine, diethanolamine,triethanolamine, dicyclohexylamine, and N,N-dibenzylethylenediamine. Thepreferred inorganic acid salt includes salts with hydrochloric acid,hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Thepreferred organic acid salt includes salts with formic acid, aceticacid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid,maleic acid, citric acid, succinic acid, malic acid, methanesulfonicacid, benzenesulfonic acid, and p-toluene sulfonic acid. The preferredsalt with a basic amino acid includes salts with arginine, lysine,ornithine, etc., while the preferred salt with an acidic amino acidincludes salts with aspartic acid and glutamic acid.

The compound of general formula (I) according to the present inventioncan be produced in accordance with the following reaction scheme.

wherein each symbol is as defined above. In this process, a compound ofthe formula (IV) [hereinafter sometimes referred to as compound (IV)] ora reactive derivative in the carboxyl thereof, or a salt thereof, isreacted with a compound of the formula (V) [hereinafter sometimesreferred to as compound (V)] or a reactive derivative thereof, or a saltthereof, to provide compound (I).

The above reaction can be carried out by the routine liquid-phase orsolid-phase (stationary) technique known to those skilled in peptidesynthesis. As to such known routine procedures and analogous procedures,the descriptions in the following literature are incorporated herein byreference: Izumiya, Nobuo et al.: Pepuchido Gosei no Kiso to Jikken(Fundamentals and Experiments in Peptide Synthesis), Maruzen, 1985;Yajima, Haruaki & Sakakibara, Shumpei: Seikagaku Jikken Koza 1(Biochemical Experiment Series 1), Japanese Biochemical Society (ed.),Tokyo Kagaku Dojin, 1977; Kimura, Toshiya: Zoku Seikagaku Jikken Koza 1(New Biochemical Experiment Series 1, Japanese Biochemical Society(ed.), Tokyo Kgaku Dojin, 1987; Suzuki, Nobuo: Jikken Kogaku Koza (4thEdition) 22, Yuki Gosei IV (Experimental Chemistry Series (Edition IV)22, Organic Synthesis IV), The Chemical Society of Japan (ed.), Maruzen,1992.

The preferred reactive derivative in the carboxyl of compound (IV)includes acid halide, acid anhydride, activated amide, and activatedester. The acid halide includes but is not limited to acid chloride. Theacid anhydride includes mixed acid anhydrides with various acids such assubstituted phosphoric acid (dialkylphosphoric acid, phenylphosphoricacid, diphenylphosphoric acid, dibenzyl phosphoric acid, halophosphoricacid, etc.), dialkylphosphorous acid, sulfurous acid, thiosulfuric acid,sulfuric acid, sulfonic acids (methanesulfonic acid, etc.), aliphaticcarboxylic acids (acetic acid, propionic acid, butyric acid, isobutyricacid, pivalic acid, pentanoic acid, isopentanoic acid, trichloroaceticacid, etc.), and aromatic carboxylic acids (benzoic acid etc.) as wellas symmetric acid anhydride. The preferred activated amide includes butis not limited to imidazole, 4-substituted imidazole, dimethylpyrazole,triazole, and tetrazole. The preferred activated ester includes but isnot limited to the cyanomethyl ester, methoxymethyl ester,dimethyliminomethyl ester, vinyl ester, propargyl ester, p-nitrophenylester, trichlorophenyl ester, pentachlorophenyl ester, methylphenylester, phenylazophenyl ester, phenylthio ester, p-nitrophenylthio ester,p-cresylthio ester, carboxymethylthio ester, pyranyl ester, pyridylester, 8-quinolylthio ester, etc. and esters with N-hydroxy compoundssuch as N,N-dimethylhydroxyamine, 1-hydroxy-2-(1H)-pyridone,N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-1H-benzotriazole,etc. The preferred salt of compound (IV) or a reactive derivativethereof includes alkali metal salts, e.g. sodium salt, potassium salt,etc., alkaline earth metal salts such as calcium salt, magnesium salt,etc., aluminum salt, and ammonium salt, as well as salts with organicbases such as trimethylamine salt, triethylamine salt, pyridine salt,picoline salt, ethanolamine salt, diethanolamine salt, triethanolaminesalt, dicyclohexylamine salt, N,N-dibenzylethylenediamine salt, etc. Thekind of reactive derivative can be selected according to the type ofcompound (IV) to be used.

The preferred reactive derivative in the amino group of compound (V)includes Schiff base type imino and enamine tautomers available onreaction of compound (V) with carbonyl compounds such as aldehydes andketones, silyl derivatives available on reaction of compound (V) withsilyl compounds such as bis(trimethylsilyl)acetamide,mono(trimethyl-silyl)acetamide, bis(trimethylsilyl)urea, etc., andderivatives available on reaction of compound (V) with phosphorustrichloride or phosgene. The preferred salts of the compound (V) and itsreactive derivative include salts with inorganic acids, such ashydrochloride, hydrobromide, nitrate, sulfate, phosphate, etc. and saltswith organic acids, such as formate, acetate, trifluoroacetate,fumarate, oxalate, tartrate, maleate, citrate, succinate, malate,methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc. Thesereactive derivatives can be selectively used according to the type ofcompound (V).

The reaction between compounds (IV) and (V) is generally conducted inwater, a common solvent, e.g. alcohol (e.g. methanol, ethanol, etc.),acetone, dioxane, acetonitrile, chloroform, methylene chloride, ethylenechloride, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, andpyridine, although the reaction can be carried out in any other organicsolvent that does not interfere with the reaction. The common organicsolvent mentioned above may be used in admixture with water. Whencompound (IV) is used either in a free form or in the form of a salt inthe above reaction, the reaction is preferably conducted in the presenceof a common condensing agent such as N,N′-dicyclohexylcarbodiimide,N-cyclohexyl-N′-morpholinoethylcarbodiimide,N-cyclohexyl-N′-(4-diethylaminocyclohexyl)carbodiimide,N,N′-diethylcarbodiimide, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide,N,N′-carbonyl-bis(2-methylimidazole),pentamethyleneketene-N-cyclohexylimine,diphenylketene-N-cyclohexylimine, ethoxyacetylene,1-alkoxy-1-chloroethylene, trimethyl phosphite, ethyl polyphosphate,isopropyl polyphosphate, phosphorus oxychloride,diphenylphosphorylazide, thionyl chloride, oxalyl chloride, haloformicacid lower alkyl esters (e.g. ethyl chloroformate, isopropylchloroformate, etc.), triphenylphosphine, N-hydroxybenzotriazole,1-(p-cholorobenzenesulfonyloxy-6-chloro-1H-benzotriazole, Vilsmeierreagents prepared by reacting N,N-dimethylformamide with thionylchloride, phosgene, trichloromethyl chloroformate, phosphorusoxychloride, or the like. The reaction may be carried out in thepresence of an inorganic or organic base, e.g. alkali metalhydrogencarbonate, tri(C₁-C₆)alkylamine, pyridine,N-(C₁-C₆)alkylmorpholine, N,N-di-(C₁-C₆)alkylbenzylamine, etc. Thereaction temperature is not so critical and the reaction can begenerally carried out under cooling, at ambient temperature, or undermild heating.

The structural formulas of the compounds synthesized in the Exampleswhich appear hereinafter are shown below.

TABLE 1

Com. No. n R¹ R² R³ R⁴ R⁶ 1 0 —COOEt H benzyl H 4-fluoro 2 0 —COOEt Hbenzyl H 2-fluoro 3 0 —COOEt H isobutyl H 4-fluoro 4 0 —COOEt H isobutylH H 7 0 —COOEt H isobutyl H 2-chloro 8 0 —COOEt H isobutyl H 3-chloro 90 —COOEt H isobutyl H 4-chloro 10 0 —COOEt H isobutyl H 4-methoxy 11 0—COOH H benzyl H 4-fluoro 12 0 —COOH H benzyl H 2-fluoro 13 0 —COOH Hisobutyl H 4-fluoro 14 0 —COOH H isobutyl H H 17 0 —COOH H isobutyl H2-chloro 18 0 —COOH H isobutyl H 3-chloro 19 0 —COOH H isobutyl H4-chloro 20 0 —COOH H isobutyl H 4-methoxy 21 0 —COOH H isopropyl H2-chloro 22 0 —COOH H H H 2-chloro 23 0 —COOH H methyl H 2-chloro 24 0—COOH H sec-butyl H 2-chloro 25 1 —COOH H H H 2-chloro 26 0 —COOH methylH H 2-chloro 27 0 —COOH pyrrolidinyl H 2-chloro 28 0 —COOH H—CH₂—S—CH₂NHCOCH₃ H 2-chloro 29 0 —COOH H —CH₂CH₂—S—CH₃ H 2-chloro 30 0—COOH H —CH₂CH₂CONH₂ H 2-chloro 31 0 —COOH H cyclopentyl 4-fluoro

TABLE 2

Com. No. n R¹ R² R³ R⁴ R⁷ 5 0 —COOEt H isobutyl H —N(CH₃)₂ 6 0 —COOEt Hisobutyl H

15 0 —COOH H isobutyl H —N(CH₃)₂ 16 0 —COOH H isobutyl H

32 0

H isobutyl H

33 0 —CONHOH H isobutyl H

As a peptide aldehyde compound having a strong cysteine proteaseinhibitory effect, a new compound of the following formula (VI) may beused,

wherein each symbol is as defined above.

Note that when the amino acid to be used in the present invention has anoptical isomer, it is an L compound unless specifically indicated.

The C₆-C₁₀ aryl at R¹¹ may be, for example, phenyl, naphthyl,pentaphenyl, indenyl and azulenyl, with preference given to phenyl andnaphthyl. The substituent optionally possessed by aryl may be, forexample, halogen atom (e.g., fluorine and chlorine), alkyl having 1 to 5carbon atoms, trifluoromethyl, alkoxy having 1 to 5 carbon atoms,hydroxy, acyloxy having 2 to 5 carbon atoms, carboxyl and acyl having 2to 5 carbon atoms, with preference given to halogen atom and alkylhaving 1 to 5 carbon atoms, and more preference given to fluorine,chlorine and methyl. Examples of preferable R¹¹ include 4-fluorophenyl,4-chlorophenyl, p-tolyl and 2-naphthyl.

The C₁-C₄ alkyl at R¹² and R¹³ may be, respectively, for example,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl ortert-butyl, with preference given to propyl, isopropyl and tert-butyl,and more preference given to isopropyl. It is preferable that one of R¹²and R¹³ be hydrogen and the other be propyl, isopropyl, isobutyl ortert-butyl, with more preference given to R¹² being propyl, isopropyl,isobutyl or tert-butyl, and R¹³ being hydrogen, with still morepreference given to R¹² being isopropyl and R¹³ being hydrogen. The ringhaving 3 to 7 carbon atoms, which is optionally formed by R¹² and R¹³may be, for example, cyclopropylidene, cyclobutylidene,cyclopentylidene, cyclohexylidene or cycloheptylidene, with preferencegiven to cyclohexylidene.

The lower alkyl at R¹⁴ may be linear, branched or cyclic alkyl having 1to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl,tert-pentyl, hexyl, 4-methylpentyl, 1,1-dimethylbutyl,2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like, withpreference given to methyl and isobutyl.

The aryl which optionally substitutes said lower alkyl may be, forexample, phenyl, 1-naphthyl or 2-naphthyl, with preference given tophenyl. The cycloalkyl which optionally substitutes said lower alkyl maybe, for example, cyclopropane, cyclobutane, cyclopentane or cyclohexane,with preference given to cyclohexane. The aromatic heterocycle whichoptionally substitutes said lower alkyl may be, for example,heteromonocyclic residue and condensed heterocyclic residue substitutedby oxygen, nitrogen or sulfur atom. Examples of heteromonocyclic residueinclude pyrolyl, furanyl, thiophenyl, oxazolyl, thiazolyl, imidazolyl,pyrazolyl, pyridyl and the like; and examples of condensed heterocyclicresidue include indolyl, quinolyl, benzothiophenyl, benzofuranyl,indazolyl, quinazolynyl, phthaladinyl, quinoxalynyl and the like, withpreference given to indolyl.

Examples of R¹⁴ include isobutyl, benzyl, cyclohexylmethyl,indol-3-ylmethyl and the like.

The salts of the compound of the formula (VI) are preferablyphysiologically acceptable ones, such as salts with inorganic base,salts with organic base, salts with inorganic acid, salts with organicacid, salts with basic or acidic amino acid, and the like. Examples ofpreferable salts with inorganic base include alkali metal salts such assodium salt and potassium salt; alkaline earth metal salts such ascalcium salt and magnesium salt; and aluminum salt and ammonium salt.Examples of preferable salts with organic base include salts withtrimethylamine, pyridine, picoline, ethanolamine, diethanolamine,triethanolamine, dicyclohexylamine, N,N-dibenzylethylenediamine and thelike. Examples of preferable salts with inorganic acid include saltswith hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid and the like. Examples of preferable salts with organicacid include salts with formic acid, acetic acid, trifluoroacetic acid,fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid,succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and the like. Examples of preferable salts withbasic amino acid include salts with arginine, lysine, ornithine and thelike, and examples of preferable salts with acidic amino acid includesalts with aspartic acid, glutamic acid and the like.

The compound (VI) can be produced, for example, by the followingreactions,

wherein each symbol is as defined above.

Sulfonyl chloride of the formula (VII) [hereinafter sometimes referredto as compound (VII)] may be, for example, naphthalenesulfonyl chloride,toluenesulfonyl chloride, fluorobenzenesulfonyl chloride,chlorobenzenesulfonyl chloride, bromobenzenesulfonyl chloride andbenzenesulfonyl chloride.

The compound of the formula (VIII) [hereinafter sometimes referred to ascompound (VIII)] may be, for example, glycine, alanine, valine,D-valine, norvaline, leucine, isoleucine, norleucine, tert-leucine,1-aminocyclopropanecarboxylic acid, 1-aminocyclobutanecarboxylic acid,1-aminocyclopentanecarboxylic acid, 1-aminocyclohexanecarboxylic acidand the like.

The reaction of compound (VII) and compound (VIII) can be carried out bya method generally known, such as Shotten-Baumann reaction.

The compound of the formula (IX) and N-hydroxysuccinimide may bedissolved in an organic solvent generally used, such as tetrahydrofuran,dichloromethane, chloroform and ethyl acetate, and condensed using acondensing agent. Examples of the condensing agent includeN,N-dicyclohexyl carbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and thelike.

The amino alcohol of the formula (XI) [hereinafter sometimes referred toas compound (XI)] may be, for example, valinol, leucinol, D-leucinol,phenylalaninol, tryptophanol or (s)-2-amino-3-cyclohexyl-1-propanol.

The compound of the formula (X) and compound (XI) are, for example,dissolved in a solvent such as tetrahydrofuran, dichloromethane,chloroform and ethyl acetate, and are reacted in the presence of a base(e.g., triethylamine and pyridine).

The compound of the formula (XII) is oxidized with an oxidizing agent(e.g., sulfur trioxide-pyridine complex, oxalyl chloride and chromicacid-pyridine) to give a new compound (VI).

While the reaction temperature is not particularly limited, the reactiongenerally proceeds under cooling, at room temperature or under heating.

The structural formula of the compounds obtained in Examples to bementioned later are shown in the following.

TABLE 3

Com. No. R¹¹ R¹² R¹⁴ * 34 2-naphthyl isopropyl isobutyl S 354-fluorophenyl isopropyl isobutyl S 36 4-chlorophenyl isopropyl isobutylS 37 4-tolyl isopropyl isobutyl S 38 2-naphthyl tert-butyl isobutyl S 404-fluorophenyl butyl isobutyl S 41 4-fluorophenyl propyl isobutyl S 422-naphthyl tert-butyl benzyl S 43 4-fluorophenyl isopropyl benzyl S 442-naphthyl isopropyl benzyl S 45 4-chlorophenyl isopropyl benzyl S 464-tolyl isopropyl benzyl S 48 4-chlorophenyl isopropyl

S 49 4-fluorophenyl isopropyl

S 51 2-naphthyl tert-butyl

S 52 4-fluorophenyl isopropyl cyclohexylmethyl S 53 2-naphthyl isopropylcyclohexylmethyl S 54 4-chlorophenyl isopropyl cyclohexylmethyl S 564-fluorophenyl isopropyl isobutyl R

TABLE 4

Com. No. R¹¹ R¹⁴ * 39 4-fluorophenyl isobutyl S 55 4-fluorophenylisobutyl R

TABLE 5

Com. No. R¹¹ R¹⁴ 47 2-naphthyl benzyl 50 2-naphthyl

Cysteine protease inhibitory compounds can be administered systemicallyor locally. For systemic administration, they are administered orally orparenterally by, for example, intravenous injection, subcutaneousinjection, intramuscular injection and the like. For localadministration, they are administered transdermally, transmucosally,intranasally, intraocularly or other route.

Cysteine protease inhibitory compounds can be used to formulatepharmaceutical compositions. Examples of compositions to be orallyadministered to human include powders, granules, tablets, capsules,syrups, liquids, and the like. When the composition is prepared intopowders, granules, tablets and the like, optional pharmaceuticalcarriers suitable for preparing solid compositions, such as vehicles(e.g., starch, glucose, fruit sugar, sucrose and the like), lubricants(e.g., magnesium stearate), disintegrators (e.g., starch and crystallinecellulose), and binders (e.g., starch and gum arabic). The compositionsmay be coated with gelatin, sucrose and the like are admixed asappropriate. When the composition is syrup or liquid, for example,stabilizers (e.g., sodium edetate), suspending agents (e.g., gum arabicand carmellose), corrigents (e.g., simple syrup and glucose), aromaticsand the like may be used as appropriate. A parenteral composition may beinjections or suppositories. When the composition is an injection, forexample, solvents (e.g., distilled water for injection), stabilizers(e.g., sodium edetate), isotonizing agents (e.g., sodium chloride,glycerine and mannitol), pH adjusting agents (e.g., hydrochloric acid,citric acid and sodium hydroxide), suspending agents (e.g., methylcellulose) and the like may be used. When the composition issuppositories, for example, a base for suppositories such as cacaobutter and macrogols, may be used as appropriate. Examples ofcompositions for external use include ointment, cream, lotion,collunarium, eye drop and the like. These compositions for external usemay contain, in addition to said inhibitory compound, for example, knowncompounds such as ointment base (e.g., petrolatum and lanolin), solvent(e.g., physiological saline and purified water), stabilizer (e.g.,sodium edetate and citric acid), wetting agent (e.g., glycerine),emulsifier (e.g., polyvinylpyrrolidone), suspending agent (e.g.,hydroxypropylmethylcellulose and methylcellulose), surfactant (e.g.,polysorbate 80 and polyoxyethylene hydrogenated castor oil),preservative (e.g., benzalkonium chloride, p-hydroxybenzoate andchlorobutanol), buffer (e.g., boric acid, sodium tetraborate, sodiumacetate, citrate buffer and phosphate buffer), isotonizing agent (e.g.,sodium chloride, glycerol and mannitol), pH adjusting agent (e.g.,hydrochloric acid and sodium hydrochloride) and the like as appropriate.

The angiogenesis inhibitor of the present invention may contain otherpharmaceutical ingredients such as antiinflammatory drug, antitumor drugand antimicrobial agent, and the like.

While the dose of the cysteine protease inhibitory compound may varydepending on target disease, symptom, administration target,administration route and the like, the amount per dose is generally1-500 mg, preferably 10-200 mg by oral administration, and generally0.1-100 mg, preferably 1-50 mg by injection. When the composition isadministered locally, for example, an eye drop adjusted generally to0.001-1.0 w/v %, preferably 0.01-0.5 w/v %, is instilled to the eye by20-50 μl at a time for 5 or 6 times a day.

The present invention is described in more detail by way of Examples andExperimental Examples in the following, which by no way limit thepresent invention.

The following Reference Examples, Examples and Experimental Examples areall intended to describe the present invention in further detail andshould by no means be construed as defining the scope of the invention.

Example 1 (Tablet)

E-64 30 mg Lactose 80 mg Starch 17 mg Magnesium stearate 3 mg

Using the above ingredients as the material for one tablet, tablets arepreared by a conventional method. Where necessary, sugar coating may beapplied.

Example 2 (Injection)

Leupeptin 100 mg Sodium chloride 900 mg 1N sodium hydroxide q.s.Distilled water for injection total 100 ml

The above ingredients are admixed by a conventional method to giveinjections.

Example 3 (Eye drop)

27 mer calpastatin peptide 1 g Boric acid 0.7 g Sodium tetraborate q.s.Sodium chloride 0.5 g Hydroxymethylcellulose 0.1 g EDTA sodium 0.02 gBenzalkonium chloride 0.005 g Sterile purified water total 100 ml

The above ingredients are admixed by a conventional method to givesuspension for instillation.

Reference Example 1

To a solution of N-tert-butoxycarbonylphenylalanine (53 g, 0.2 mol) andp-nitrophenol (27.8 g, 0.2 mol) in ethyl acetate (200 ml) in anice-water bath was dropwise added a solution ofN,N′-dicyclohexylcarbodiimide (41.2 g, 0.2 mol) in ethyl acetate (100ml), and the mixture was stirred in an ice-water bath for 3 hours andthen at room temperature for 20 hours. The precipitatedN,N′-dicyclohexylcarbodiurea was filtered off and the filtrate wasconcentrated under reduced pressure. The residue thus obtained wasrecrystallized from ethyl acetate-hexane to giveN-tert-butoxycarbonylphenylalanine p-nitrophenyl ester [61.7 g, 80% (%by weight, hereinafter the same)].

Reference Example 2

To a solution of N-tert-butoxycarbonylleucine (6.94 g, 30 mmol) andN-hydroxysuccinimide (3.45 g, 30 mmol) in dioxane (50 ml) in anice-water bath was dropwise added a solution ofN-ethyl-N′-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (5.75 g,30 mmol) in dioxane, and the mixture was stirred in an ice-water bathfor 20 minutes and then at room temperature for 24 hours. The reactionmixture was poured into cold water and extracted with ethyl acetate. Theextract was washed with 10 w/v % aqueous citric acid solution, 10 w/v %aqueous sodium hydrogencarbonate solution, and saturated brine in theorder mentioned, and the organic layer was dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. The residuewas recrystallized from isopropyl ether to giveN-tert-butoxycarbonylleucine N-hydroxysuccinimide ester (7.77 g, 78.9%).

Reference Example 3

To a solution of 1-(4-fluorophenyl)piperazine dihydrochloride (2.53 g,10 mmol) in N,N-dimethylformamide (40 ml) were added triethylamine (2.8ml, 20 mmol) and N-tert-butoxycarbonylphenylalanine p-nitrophenyl ester(2.65 g, 10 mmol) in the order mentioned, and the mixture was stirred atroom temperature overnight. The reaction mixture was poured into coldwater and extracted with ethyl acetate. The extract was washed with 1w/v % aqueous ammonia, saturated brine, 0.1N hydrochloric acid,saturated brine, saturated aqueous sodium hydrogencarbonate solution,and saturated brine in the order mentioned, and the organic layer wasdried over anhydrous magnesium sulfate and further concentrated underreduced pressure. The residue was purified by silica gel columnchromatography using chloroform-methanol (50:1, v/v) to give1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate(2.7 g, 92.2%) as colorless oil.

Reference Example 4

Using 1-(o-fluorophenyl)piperazine monohydrochloride instead of1-(4-fluorophenyl)piperazine dihydrochloride, the procedure of ReferenceExample 3 was repeated to give1,1-dimethylethyl-2-(4-(2-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate(1.89 g, 88.4%).

Reference Example 5

To a solution of 1-(4-fluorophenyl)piperazine dihydrochloride (0.91 g, 3mmol) and N-tert-butoxycarbonylleucine N-hydroxysuccinimide ester (0.99g, 3 mmol) in dichloromethane (50 ml) was added triethylamine (1.3 ml, 9mmol), and the mixture was stirred at room temperature for 20 hours. Thereaction mixture was washed with 0.1N hydrochloric acid, saturatedaqueous sodium hydrogencarbonate solution, water, and saturated brine inthe order mentioned, and the organic layer was dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. The residuewas subjected to silica gel column chromatography using ethylacetate-hexane (1:1, v/v) to give 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamate(1.05 g, 89.0%) as colorless oil.

Reference Example 6

Using 4-phenylpiperazine instead of 1-(4-fluorophenyl)piperazinedihydrochloride, the procedure of Reference Example 5 was repeated togive 1,1-dimethylethyl2-(4-phenyl-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamate (7.99g, 99%).

Reference Example 7

Using 1-dimethylsulfamoylpiperazine instead of1-(4-fluorophenyl)-piperazine dihydrochloride, the procedure ofReference Example 5 was repeated to give 1,1-dimethylethyl2-(4-dimethylsulfamoyl-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamate(7.19 g, 88.4%).

Reference Example 8

Using p-toluenesulfonylpiperazine instead of1-(4-fluorophenyl)-piperazine dihydrochloride, the procedure ofReference Example 5 was repeated to give 1,1-dimethylethyl2-(4-(4-methylphenylsulfonyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamate(6.95 g, 79.4%).

Reference Example 9

Using 1-(2-chlorophenyl)piperazine instead of1-(4-fluorophenyl)-piperazine dihydrochloride, the procedure ofReference Example 5 was repeated to give 1,1-dimethylethyl2-(4-(2-chlorophenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamate(5.70 g, 95.5%).

Reference Example 10

Using 1-(m-chlorophenyl)piperazine monohydrochloride instead of1-(4-fluorophenyl)piperazine dihydrochloride, the procedure of ReferenceExample 5 was repeated to give 1,1-dimethylethyl2-(4-(3-chlorophenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamate(2.63 g, 88.4%).

Reference Example 11

Using 1-(4-chlorophenyl)piperazine monohydrochloride instead of1-(4-fluorophenyl)piperazine dihydrochloride, the procedure of ReferenceExample 5 was repeated to give 1,1-dimethylethyl2-(4-(4-chlorophenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamate(2.83 g, 94.8%).

Reference Example 12

Using N-(p-methoxyphenyl)piperazine succinate instead of1-(4-fluorophenyl)piperazine dihydrochloride, the procedure of ReferenceExample 5 was repeated to give 1,1-dimethylethyl2-(4-(4-methoxyphenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamate(2.73 g, 92.3%).

Reference Example 13

To a solution of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate(2.7 g, 6.3 mmol) in ethyl acetate (20 mmol) under ice-cooling wasdropwise added 4N HCl/ethyl acetate (20 ml), and the mixture was stirredat room temperature overnight. The resulting crystals were recovered byfiltration and recrystallized from ethanol-diethyl ether to give1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride (2.2 g, 96.1%) as pale-yellow crystals.

Reference Example 14

Using 1,1-dimethylethyl2-(4-(2-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-1-oxo-3-phenylpropyl)-4-(2-fluorophenyl)piperazinehydrochloride (1.3 g, 99.1%) as white crystals.

Reference Example 15

Using 1,1-dimethylethyl2-(4-(2-fluorophenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-4-methyl-1-oxopentyl)-4-(4-fluorophenyl)piperazinehydrochloride (0.56 g, 70.4%) as white crystals.

Reference Example 16

Using 1,1-dimethylethyl2-(4-phenyl-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-4-methyl-1-oxopentyl)-4-phenylpiperazine hydrochloride (6.5g, 99.2%) as white crystals.

Reference Example 17

Using 1,1-dimethylethyl2-(4-dimethylsulfamoyl-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-4-methyl-1-oxopentyl)-4-dimethylsulfamoylpiperazinehydrochloride (5.0 g, 83.3%) as white crystals.

Reference Example 18

Using 1,1-dimethylethyl2-(4-(4-methylphenylsulfonyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)-ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-4-methyl-1-oxopentyl)-4-(4-methylphenylsulfonyl)-piperazinehydrochloride (4.83 g, 78.4%) as white crystals.

Reference Example 19

Using 1,1-dimethylethyl2-(4-(2-chlorophenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-4-methyl-1-oxopentyl)-4-(2-chlorophenyl)piperazinehydrochloride (1.54 g, 62.6%) as white crystals.

Reference Example 20

Using 1,1-dimethylethyl2-(4-(3-chlorophenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-4-methyl-1-oxopentyl)-4-(3-chlorophenyl)piperazinehydrochloride (1.40 g, 65.7%) as white crystals.

Reference Example 21

Using 1,1-dimethylethyl2-(4-(4-chlorophenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-4-methyl-1-oxopentyl)-4-(4-chlorophenyl)piperazinehydrochloride (1.50 g, 65.3%) as white crystals.

Reference Example 22

Using 1,1-dimethylethyl2-(4-(4-methoxyphenyl)-1-piperazinyl)-2-oxo-1-(2-methylpropyl)ethylcarbamateinstead of 1,1-dimethylethyl2-(4-(4-fluorophenyl)-1-piperazinyl)-2-oxo-1-(phenylmethyl)ethylcarbamate,the procedure of Reference Example 13 was repeated to give1-(2-amino-4-methyl-1-oxopentyl)-4-(4-methoxyphenyl)piperazinehydrochloride (2.21 g, 87.4%) as white crystals.

Reference Example 23

To a solution of N-tert-butoxycarbonyl-L-valine (2.27 g, 10 mmol) and1-(2-chlorophenyl)piperazine (2.00 g, 10 mmol) in N,N-dimethylformamide(50 ml) under ice-cooling was dropwise added a solution of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.2 g, 11mmol) and 1-hydroxybenzotriazole (1.5 g, 11 mmol) in dichloromethane (50ml), and the reaction mixture was stirred at room temperature for 15hours. The dichloromethane was then distilled off under reduced pressureand ethyl acetate (200 ml) was added to the residue. The ethyl acetatelayer was washed successively with 10 w/v % aqueous citric acidsolution, saturated aqueous sodium hydrogen carbonate solution, andsaturated brine in the order mentioned and dried over anhydrous sodiumsulfate. The solvent was then distilled off and the residue wassubjected to silica gel chromatography. Elution with ethylacetate-n-hexane (1:2, v/v) gave 1,1-dimethylethyl2-(4-(2-chlorophenyl)-piperazinyl)-2-oxo-(s)-1-(2-propyl)ethylcarbamate.This colorless oil was dissolved in ethyl acetate (50 ml), and 4NHCl/ethyl acetate (50 ml) was added dropwise under ice-cooling. Themixture was stirred at room temperature for 3 hours. The reactionproduct was filtered and washed with ethyl acetate-n-hexane (1:1, v/v)to give 1-((s)-2-amino-3-methyl-1-oxobutyl)-4-(2-chlorophenyl)piperazinehydrochloride (3.32 g, 95.8%) as colorless crystals.

Reference Example 24

Starting with N-tert-butoxycarbonylglycine, the procedure of ReferenceExample 23 was repeated to give1-(2-amino-1-oxoethyl)-4-(2-chlorophenyl)piperazine hydrochloride (2.4g, 90.4%) as colorless crystals.

Reference Example 25

Starting with N-tert-butoxycarbonyl-L-alanine, the procedure ofReference Example 23 was repeated to give1-((s)-2-amino-1-oxopropyl)-4-(2-chlorophenyl)piperazine hydrochloride(1.7 g, 58.8%) as colorless crystals.

Reference Example 26

Starting with N-tert-butoxycarbonyl-L-isoleucine, the procedure ofReference Example 23 was repeated to give1-((s)-2-amino-3-methyl-1-oxo-pentyl)-4-(2-chlorophenyl)piperazinehydrochloride (3.4 g, 90.2%) as colorless crystals.

Reference Example 27

Starting with N-tert-butoxycarbonyl-β-alanine, the procedure ofReference Example 23 was repeated to give1-(3-amino-1-oxopropyl)-4-(2-chlorophenyl)piperazine hydrochloride (2.9g, 90.0%) as colorless crystals.

Reference Example 28

Starting with N-tert-butoxycarbonylsarcosine, the procedure of ReferenceExample 23 was repeated to give1-(2-methylamino-1-oxoethyl)-4-(2-chlorophenyl)piperazine hydrochloride(3.0 g, 93.3%) as colorless crystals.

Reference Example 29

Starting with N-tert-butoxycarbonyl-L-proline, the procedure ofReference Example 23 was repeated to give1-(1-(2-pyrrolidinyl)-1-oxomethyl)-4-(2-chlorophenyl)piperazinehydrochloride (4.3 g, 98.0%) as colorless crystals.

Reference Example 30

Starting with N-tert-butoxycarbonyl-(s)-acetamidomethyl)-L-cysteine, theprocedure of Reference Example 23 was repeated to give1-((s)-2-amino-3-(acetylaminomethylthio)-1-oxopropyl)-4-(2-chlorophenyl)-piperazinehydrochloride (4.0 g, 95.9%) as colorless crystals.

Reference Example 31

Starting with N-tert-butoxycarbonyl-L-methionine, the procedure ofReference Example 23 was repeated to give1-((s)-2-amino-4-methylthio-1-oxo-butyl)-4-(2-chlorophenyl)piperazinehydrochloride (3.7 g, 97.1%) as colorless crystals.

Reference Example 32

Starting with N-tert-butoxycarbonyl-L-glutamine, the procedure ofReference Example 23 was repeated to give1-((s)-2-amino-4-carbamoyl-1-oxo-butyl)-4-(2-chlorophenyl)piperazinehydrochloride (2.7 g, 60.7%) as colorless crystals.

Example 4

To a solution of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride (1.82 g, 5 mmol) in N,N-dimethylformamide (20 ml) wasadded triethylamine (0.697 ml, 5 mmol), and the mixture was stirred atroom temperature for 10 minutes. To the reaction mixture was added ethylp-nitrophenyl L-trans-epoxysuccinate (1.41 g, 5 mmol), synthesized inaccordance with the method of Tamai et al. [Chem. Pharm. Bull., 35, 1098(1987)], and the mixture was stirred at room temperature for 20 hours.The reaction mixture was poured into cold water and extracted with ethylacetate. The extract was washed successively with 1 w/v % aqueousammonia, saturated brine, 0.1N hydrochloric acid, saturated brine,saturated aqueous sodium hydrogencarbonate solution, and saturated brinein the order mentioned, and the organic layer was dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. The residuewas subjected to silica gel column chromatography, elution being carriedout with ethyl acetatehexane (1:1, v/v) to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperadinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate(1.2 g, 51.1%; Compound 1).

¹H NMR (CDCl₃) δ: 1.31 (t, 3H, J=7.0 Hz, —C—CH₃), 2.42-2.50 (m, 1H,piperazine ring), 2.83-2.94 (m, 2H, piperazine ring), 3.00 (d, 2H, J=7.6Hz, ph-CH₂—C—), 2.97-3.07 (m, 1H, piperazine ring), 3.14-3.22 (m, 1H,piperazine ring), 3.35 (d, 1H, J=1.9 Hz, epoxy ring), 3.43-3.52 (m, 1H,piperazine ring), 3.64 (d, 1H, J=1.9 Hz, epoxy ring), 3.71 (t, 2H, J=5.4Hz, piperazine ring), 4.25 (dq, 2H, J=7.3, 2.6 Hz, —O—-CH₂—C), 5.18 (q,1H, J=5.3 Hz, —N—CH—CO), 6.75-6.83 (m, 2H, aromatic), 6.91-7.04 (m, 2H,aromatic, 1H, NH), 7.17-7.34 (m, 5H, aromatic).

Example 5

Using 1-(2-amino-1-oxo-3-phenylpropyl)-4-(2-fluorophenyl)piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate(0.83 g, 58.6%; Compound 2).

¹H NMR (CDCl₃) δ: 1.31 (t, 3H, J=7.1 Hz, —C—CH₃), 2.42-2.49 (m, 1H,piperazine ring), 2.72-2.90 (m, 2H, piperazine ring), 3.02 (d, 2H, J=8.3Hz, ph-CH₂—C—), 2.94-3.10 (m, 1H, piperazine ring), 3.17-3.29 (m, 1H,piperazine ring), 3.36 (d, 1H, J=1.7 Hz, epoxy ring), 3.44-3.57 (m, 1H,piperazine ring), 3.65 (d, 1H, J=1.3 Hz, epoxy ring), 3.66-3.80 (m, 2H,piperazine ring), 4.25 (dq, 2H, J=7.1, 2.3 Hz, —O—CH₂—C), 5.19 (q, 1H,J=7.6 Hz, —N—CH—CO), 6.8 (t, 1H, J=8.3 Hz, —NH—), 6.93-7.11 (m, 4H,aromatic), 7.18-7.34 (m, 5H, aromatic).

Example 6

Using 1-(2-amino-4-methyl-1-oxopentyl)-4-(4-fluorophenyl)piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylate(0.30 g, 45.6%; Compound 3).

hu 1H NMR (CDCl₃) δ: 0.93 (d, 3H, J=6.3 Hz, —C—CH₃), 1.00 (d, 3H, J=6.3Hz, —C—CH₃), 1.32 (t, 3H, J=7.3 Hz, —C—CH₃), 1.40-1.63 (m, 3H,—C—CH₂—CH—C₂), 3.06-3.15 (m, 4H, piperazine ring), 3.49 (d, 1H, J=1.7Hz, epoxy ring), 3.60-3.89 (m, 4H, piperazine ring), 3.68 (d, 1H, J=1.7Hz, epoxy ring), 4.26 (dq, 2H, J=7.3, 3.3 Hz, —O—CH₂—C), 5.03 (dt, 1H,J=8.91, 4.3 Hz, —N—CH—CO), 6.85-7.03 (m, 5H, aromatic and —NH).

Example 7

Using 1-(2-amino-4-methyl-1-oxopentyl)-4-phenylpiperazine hydrochlorideinstead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)-piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[(4-phenyl-1-piperazinyl)carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylate(3.96 g, 63.3%; Compound 4).

¹H NMR (CDCl₃) δ: 0.93 (d, 3H, J=6.3 Hz, —C—CH₃), 1.00 (d, 3H, J=6.3 Hz,—C—CH₃), 1.32 (t, 3H, J=7.3 Hz, —C—CH₃), 1.40-1.63 (m, 3H, —C—CH₂—CH—C₂,3.16-3.24 (m, 4H, piperazine ring), 3.49 (d, 1H, J=1.7 Hz, epoxy ring),3.60-3.89 (m, 4H, piperazine ring), 3.68 (d, 1H, J=1.7 Hz, epoxy ring),4.26 (dq, 2H, J=7.3, 3.3Hz, —O—CH₂—C), 5.03 (dt, 1H, J=8.9, 4.3 Hz,—N—CH—CO), 6.90-6.95 (m, 4H, aromatic and —NH), 7.25-7.33 (m, 2H,aromatic).

Example 8

Using 1-(2-amino-4-methyl-1-oxopentyl)-4-dimethylsulfamoylpiperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[(4-dimethylsulfamoyl-1-piperazinyl)carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylate(3.7 g, 82.6%; Compound 5).

¹H NMR (CDCl₃) δ: 0.92 (d, 3H, J=6.3 Hz, —C—CH₃), 0,89 (d, 3H, J=6.3 Hz,—C—CH₃), 1.32 (t, 3H, J=7.3 Hz, —C—CH₃), 1.38-1.60 (m, 3H,—C—CH₂—CH—C₂), 2.85 (s, 6H, —N—CH₃), 3.15-3.38 (m, 4H, piperazine ring),3.48 (d, 1H, J=1.7 Hz, epoxy ring), 3.52-3.68 (m, 3H, piperazine ring),3.67 (d, 1H, J=1.7 Hz, epoxy ring), 3.79-3.87 (m, 1H, piperazine ring),4.27 (dq, 2H, J=7.3, 4.0 Hz, —O—CH₂—C), 4.96 (dt, 1H, J=8.9, 4.3 Hz,—N—CH—CO), 6.90 (d, 1H, J=8.6 Hz, —NH—).

Example 9

Using1-(2-amino-4-methyl-1-oxopentyl)-4-(4-methylphenylsulfonyl)-piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(2s)-1-[[4-(4-methylphenylsulfonyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylate(4.31 g, 95.1%; Compound 6).

¹H NMR (CDCl₃) δ: 0.87 (d, 3H, J=6.3 Hz, —C—CH₃), 0.94 (d, 3H, J=6.3 Hz,—C—CH₃), 1.30 (t, 3H, J=7.3 Hz, —C—CH₃), 1.31-1.55 (m, 3H,—C—CH₂—CH—C₂), 2.45 (s, 6H, -ph-CH₃), 2.70-2.84 (m, 2H, piperazinering), 3.22-3.54 (m, 4H, piperazine ring), 3.43 (d, 1H, J=1.7 Hz, epoxyring), 3.61 (d, 1H, J=2.0 Hz, epoxy ring), 3.68-3.78 (m, 1H, piperazinering), 3.96-4.06 (m, 1H, piperazine ring), 4.25 (dq, 2H, J=7.3, 4.0 Hz,—O—CH₂—C), 4.87 (dt, 1H, J=9.2, 4.0 Hz, —N—CH—CO), 6.81 (d, 1H, J=8.6Hz, —NH—), 7.35 (d, 2H, J=7.9 Hz, aromatic), 7.63 (d, 2H, J=8.3 Hz,aromatic).

Example 10

Using 1-(2-amino-4-methyl-1-oxopentyl)-4-(2-chlorophenyl)piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylate(0.65 g, 35.9%; Compound 7).

¹H NMR (CDCl₃) δ: 0.93 (d, 3H, J=6.3 Hz, —C—CH₃), 1.01 (d, 3H, J=6.3 Hz,—C—CH₃), 1.32 (t, 3H, J=7.3 Hz, —C—CH₃), 1.41-1.61 (m, 3H,—C—CH₂—CH—C₂), 2.96-3.10 (m, 4H, piperazine ring), 3.49 (d, 1H, J=2.0Hz, epoxy ring), 3.61-3.81 (m, 3H, piperazine ring), 3.68 (d, 1H, J=2.0Hz, epoxy ring), 3.90-3.98 (m, 1H, piperazine ring), 4.27 (dq, 2H,J=7.3, 4.0 Hz, —O—CH₂—C), 5.03 (dt, 1H, J=8.9, 4.3 Hz, —N—CH—CO), 6.91(d, 1H, J=8.6 Hz, —NH—), 7.00-7.06 (m, 2H, aromatic), 7.21-7.27 (m, 1H,aromatic), 7.37-7.41 (m, 1H, aromatic).

Example 11

Using 1-(2-amino-4-methyl-1-oxopentyl)-4-(3-chlorophenyl)piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(3-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylate(1.24 g, 68.4%; Compound 8).

¹H NMR (CDCl₃) δ: 0.93 (d, 3H, J=6.3 Hz, —C—CH₃), 1.00 (d, 3H, J=6.3 Hz,—C—CH₃), 1.32 (t, 3H, J=7.3 Hz, —C—CH₃), 1.42-1.63 (m, 3H,—C—CH₂—CH—C₂), 3.17-3.25 (m, 4H, piperazine ring), 3.48 (d, 1H, J=2.0Hz, epoxy ring), 3.60-3.90 (m, 4H, piperazine ring), 3.67 (d, 1H, J=2.0Hz, epoxy ring), 4.27 (dq, 2H, J=7.3, 4.0 Hz, —O—CH₂—C), 5.02 (dt, 1H,J=8.9, 4.3 Hz, —N—CH—CO), 6.77-6.81 (m, 1H, aromatic), 6.86-6.89 (m, 3H,aromatic and —NH), 7.16-7.22 (m, 1H, aromatic).

Example 12

Using 1-(2-amino-4-methyl-1-oxopentyl)-4-(4-chlorophenyl)piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylate(0.55 g, 27.1%; Compound 9).

¹H NMR (CDCl₃) δ: 0.93 (d, 3H, J=6.3 Hz, —C—CH₃), 1.0 (d, 3H, J=6.3 Hz,—C—CH₃), 1.32 (t, 3H, J=7.3 Hz, —C—CH₃), 1.42-1.63 (m, 3H,—C—CH₂—CH—C₂), 3.12-3.20 (m, 4H, piperazine ring), 3.48 (d, 1H, J=2.0Hz, epoxy ring), 3.60-3.90 (m, 4H, piperazine ring), 3.67 (d, 1H, J=2.0Hz, epoxy ring), 4.27 (dq, 2H, J=7.3, 4.0 Hz, —O—CH₂—C), 5.02 (dt, 1H,J=8.9, 4.3 Hz, —N—CH—CO), 6.83-6.87 (m, 2H, aromatic), 6.90 (d, 1H,J=9.9 Hz, —NH), 7.21-7.3 (m, 2H, aromatic).

Example 13

Using 1-(2-amino-4-methyl-1-oxopentyl)-4-(4-methoxyphenyl)-piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-methoxyphenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylate(0.94 g, 65.2%; Compound 10).

¹H NMR (CDCl₃) δ: 0.92 (d, 3H, J=6.6 Hz, —C—CH₃), 1.00 (d, 3H, J=6.3 Hz,—C—CH₃), 1.32 (t, 3H, J=7.3 Hz, —C—CH₃), 1.40-1.60 (m, 3H,—C—CH₂—CH—C₂), 3.03-3.11 (m, 4H, piperazine ring), 3.49 (d, 1H, J=2.0Hz, epoxy ring), 3.60-3.88 (m, 4H, piperazine ring), 3.67 (d, 1H, J=2.0Hz, epoxy ring), 3.78 (s, 3H, —O—CH₃), 4.27 (dq, 2H, J=7.3, 4.0 Hz,—O—CH₂—C), 5.03 (dt, 1H, J=8.9, 4.3 Hz, —N—CH—CO), 6.83-6.96 (m, 5H,aromatic and —NH).

Example 14

Using 1-((s)-2-amino-3-methyl-1-oxobutyl)-4-(2-chlorophenyl)-piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-methyl]propyl]amino]carbonyl]oxiranecarboxylate(1.3 g, 57.4%) as colorless oil.

Example 15

Using 1-(2-amino-1-oxoethyl)-4-(2-chlorophenyl)piperazine hydrochlorideinstead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)-piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-methyl]amino]carbonyl]oxiranecarboxylate(1.75 g, 53.5%) as colorless oil.

Example 16

Using 1-((s)-2-amino-1-oxopropyl)-4-(2-chlorophenyl)piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]ethyl]amino]carbonyl]oxiranecarboxylate(1.23 g, 55.0%) as colorless oil.

Example 17

Using 1-((s)-2-amino-3-methyl-1-oxopentyl)-4-(2-chlorophenyl)-piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-methyl]butyl]amino]carbonyl]oxiranecarboxylate(1.48 g, 56.6%) as colorless oil.

Example 18

Using 1-(3-amino-1-oxopropyl)-4-(2-chlorophenyl)piperazine hydrochlorideinstead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)-piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[2-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-ethyl]amino]carbonyl]oxiranecarboxylate(1.16 g, 52.9%) as colorless oil.

Example 19

Using 1-(2-methylamino-1-oxoethyl)-4-(2-chlorophenyl)piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[N-[[4-(2-chlorophenyl)-1-piperazinyl]-carbonyl]methyl]-N-methyl]amino]carbonyl]oxiranecarboxylate(1.55 g, 76.7%) as colorless oil.

Example 20

Using 1-(1-(2-pyrrolidinyl)-1-oxomethyl)-4-(2-chlorophenyl)-piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[(2s)-2-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-1-pyrrolidinyl]carbonyl]oxiranecarboxylate(1.42 g, 49.9%) as colorless oil.

Example 21

Using1-((s)-2-amino-3-(acetylaminomethylthio)-1-oxopropyl)-4-(2-chlorophenyl)piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-acetylaminomethylthio]ethyl]-amino]carbonyl]oxiranecarboxylate(1.19 g, 43.7%) as colorless oil.

Example 22

Using1-((s)-2-amino-4-methylthio-1-oxobutyl)-4-(2-chlorophenyl)-piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-4-methylthio]butyl]amino]carbonyl]oxiranecarboxylate(1.54 g, 61.5%) as colorless oil.

Example 23

Using1-((s)-2-amino-4-carbamoyl-1-oxobutyl)-4-(2-chlorophenyl)-piperazinehydrochloride instead of1-(2-amino-1-oxo-3-phenylpropyl)-4-(4-fluorophenyl)piperazinehydrochloride, the procedure of Example 4 was repeated to give ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-3-carbamoyl]propyl]amino]carbonyl]oxiranecarboxylate(0.2 g, 5.8%) as colorless oil.

Example 24

To a solution of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate(0.5 g, 1.06 mmol) in ethanol (20 ml) was added 0.1N sodiumhydroxide/ethanol (16 ml) under ice-cooling and the mixture was stirredat room temperature for 20 hours. The reaction mixture was poured intocold water and acidified with 1N hydrochloric acid and the resultingwhite precipitate was recovered by filtration and dried. Thisprecipitate was recrystallized from ethyl acetate-hexane to give(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylicacid (0.36 g, 77.8%; Compound 11).

¹H NMR (CDCl₃) δ: 2.34-2.41 (m, 1H, piperazine ring), 2.82-2.96 (m, 2H,piperazine ring), 2.99-3.08 (m, 1H, piperazine ring), 3.06 (d, 2H, J=7.3Hz, ph-CH₂—C—), 3.16-3.24 (m, 1H, piperazine ring), 3.49-3.58 (m, 1H,piperazine ring), 3.55 (d, 1H, J=1.7 Hz, epoxy ring), 3.57 (d, 1H, J=1.7Hz, epoxy ring), 3.71 (t, 2H, J=5.1 Hz, piperazine ring), 4.5-6.0 (brd,1H, —COOH), 5.23 (q, 1H, J=7.9 Hz, —N—CH—CO), 6.74-6.82 (m, 2H,aromatic), 6.91-7.00 (m, 2H, aromatic), 7.20-7.35 (m, 5H, aromatic),8.23 (d, 1H, J=8.6 Hz, —NH—).

Example 25

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(2-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]-oxiranecarboxylicacid (0.1 g, 29.6%; Compound 12).

¹H NMR (CDCl₃) δ: 2.38-2.43 (m, 1H, piperazine ring), 2.83-2.93 (m, 2H,piperazine ring), 2.95-3.08 (m, 1H, piperazine ring), 3.06 (d, 2H, J=7.6Hz, ph-CH₂—C—), 3.20-3.28 (m, 1H, piperazine ring), 3.49-3.66 (m, 1H,piperazine ring), 3.55 (d, 1H, J=1.7 Hz, epoxy ring), 3.58 (d, 1H, J=1.3Hz, epoxy ring), 3.67-3.80 (m, 2H, piperazine ring), 4.0-6.0 (brd, 1H,—COOH), 5.23 (q, 1H, J=7.9 Hz, —N—CH—CO), 6.77-6.87 (m, 1H, aromatic),6.93-7.09 (m, 3H, aromatic), 7.20-7.36 (m, 5H, aromatic), 8.23 (d, 1H,J=8.6 Hz, —NH—).

Example 26

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]-oxiranecarboxylicacid (0.13 g, 69.5%; Compound 13).

¹H NMR (CDCl₃) δ: 0.96 (d, 3H, J=6.6 Hz, —C—CH₃), 0.99 (d, 3H, J=6.59Hz, —C—CH₃), 1.42 (ddd, 1H, J=14.1, 10.5, 3.6 Hz, —C—CH₂—C), 1.6-1.82(m, 1H, —C—CH—C₂), 1.69 (ddd, 1H, J=14.5, 10.7, 4.23 Hz, —C—CH₂—C),3.08-3.26 (m, 4H, piperazine ring), 3.55 (d, 1H, J=1.7 Hz, epoxy ring),3.60-3.92 (m, 4H, piperazine ring), 3.62 (d, 1H, J=1.8 Hz, epoxy ring),5.08 (ddd, 1H, J=10.6, 8.6, 3.6 Hz, —N—CH—CO), 5.2-6.4 (brd, 1H, —COOH),6.84-7.03 (m, 4H, aromatic), 8.18 (d, 1H, J=8.6 Hz, —NH—).

Example 27

Using ethyl(2s,3s)-3-[[[[(1s)-1-[(4-phenyl-1-piperazinyl)-carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[(4-phenyl-1-piperazinyl)carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylicacid (1.06 g, 29.1%; Compound 14).

¹H NMR (CDCl₃) δ: 0.96 (d, 3H, J=6.6 Hz, —C—CH₃), 0.99 (d, 3H, J=6.26Hz, —C—CH₃), 1.37-1.47 (m, 1H, —C—CH—C₂), 1.64-1.80 (m, 2H, —C—CH₂—C—),3.17-3.36 (m, 4H, piperazine ring), 3.55 (d, 1H, J=1.7 Hz, epoxy ring),3.62 (d, 1H, J=1.3 Hz, epoxy ring), 3.70-3.90 (m, 4H, piperazine ring),5.10 (m, 1H, —N—CH—CO), 6.5-7.5 (brd, 1H, —COOH), 6.87-6.96 (m, 3H,aromatic), 7.27-7.33 (m, 2H, aromatic), 8.20 (d, 1H, J=8.6 Hz, —NH—).

Example 28

Using ethyl(2s,3s)-3-[[[[(1s)-1-[(4-dimethylsulfamoyl-1-piperazinyl)carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate, the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[(4-dimethylsulfamoyl-1-piperazinyl)carbonyl]-3-methyl]butyl]amino]-carbonyl]oxiranecarboxylicacid (1.28 g, 39.0%; Compound 15).

¹H NMR (CDCl₃) δ: 0.94 (d, 3H, J=6.3 Hz, —C—CH₃), 0.96 (d, 3H, J=5.6 Hz,—C—CH₃), 1.36-1.44 (m, 1H, —C—CH—C₂), 1.61-1.68 (m, 2H, —C—CH₂—C—), 2.85(s, 6H, —N—CH₃), 3.17-3.35 (m, 4H, piperazine ring), 3.48-3.60 (m, 2H,piperazine ring), 3.58 (d, 1H, J=1.7 Hz, epoxy ring), 3.62 (d, 1H, J=1.7Hz, epoxy ring), 3.70-3.80 (m, 1H, piperazine ring), 3.83-3.95 (m, 1H,piperazine ring), 4.95-5.05 (m, 1H, —N—CH—CO), 7.7-8.1 (brd, 1H, —COOH),7.94 (d, 1H, J=8.6 Hz, —NH—).

Example 29

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-methylphenylsulfonyl)-1-piperazinyl]carbonyl]-3-methyl]-butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-1-[[[[(1s)[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(4-methylphenylsulfonyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]-carbonyl]oxiranecarboxylicacid (2.8 g, 70.0%; Compound 16).

¹H NMR (CDCl₃) δ: 0.90 (d, 3H, J=6.6 Hz, —C—CH₃, 0.93 (d, 3H, J=6.6 Hz,—C—CH₃), 1.23-1.33 (m, 1H, —C—CH—C₂), 1.53-1.67 (m, 2H, —C—CH₂—C—), 2.45(s, 3H, -ph-CH₃), 2.73-2.91 (m, 2H, piperazine ring), 3.28-3.59 (m, 4H,piperazine ring), 3.45 (d, 1H, J=1.7 Hz, epoxy ring), 3.48 (d, 1H, J=1.7Hz, epoxy ring), 3.70-3.83 (m, 1H, piperazine ring), 3.98-4.08 (m, 1H,piperazine ring), 4.85-4.97 (m, 1H, —N—CH—CO), 7.35 (d, 2H, J=7.9 Hz,aromatic), 7.63 (d, 2H, J=8.3 Hz, aromatic), 7.97 (d, 1H, J=8.6 Hz,—NH—).

Example 30

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]-oxiranecarboxylicacid (0.41 g, 67.2%; Compound 17).

¹H NMR (CDCl₃) δ: 0.97 (d, 3H, J=6.9 Hz, —C—CH₃), 0.98 (d, 3H, J=7.3 Hz,—C—CH₃), 1.38-1.47 (m, 1H, —C—CH—C₂), 1.65-1.77 (m, 2H, —C—CH₂—C—),2.98-3.19 (m, 4H, piperazine ring), 3.55 (d, 1H, J=1.7 Hz, epoxy ring),3.61-3.77 (m, 2H, piperazine ring), 3.64 (d, 1H, J=1.7 Hz, epoxy ring),3.77-3.89 (m, 1H, piperazine ring), 3.89-4.15 (m, 1H, piperazine ring),5.04-5.18 (m, 1H, —N—CH—CO), 7.00-7.06 (m, 2H, aromatic), 7.21-7.28 (m,1H, aromatic), 7.37-7.41 (m, 1H, aromatic), 8.25 (d, 1H, J=8.9 Hz,—NH—).

Example 31

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(3-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(3-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]-oxiranecarboxylicacid (0.39 g, 67.2%; Compound 18).

¹H NMR (CDCl₃) δ: 0.96 (d, 3H, J=6.6 Hz, —C—CH₃), 0.99 (d, 3H, J=6.6 Hz,—C—CH₃), 1.36-1.47 (m, 1H, —C—CH—C₂), 1.64-1.80 (m, 2H, —C—CH2—C—),3.18-3.36 (m, 4H, piperazine ring), 3.53 (d, 1H, J=1.7 Hz, epoxy ring),3.60-3.92 (m, 4H, piperazine ring), 3.62 (d, 1H, J=1.7 Hz, epoxy ring),5.04-5.12 (m, 1H, —N—CH—CO), 5.5-6.5 (brd, 1H, —COOH), 6.77-6.82 (m, 1H,aromatic), 6.88-6.90 (m, 2H, aromatic), 7.17-7.23 (m, 1H, aromatic),8.21 (d, 1H, J=8.6 Hz, —NH—).

Example 32

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(4-chlorophenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]-oxiranecarboxylicacid (0.33 g, 63.4%; Compound 19).

¹H NMR (CDCl₃) δ: 0.96 (d, 3H, J=6.6 Hz, —C—CH₃), 0.99 (d, 3H, J=6.59Hz, —C—CH₃), 1.36-1.47 (m, 1H, —C—CH—C₂), 1.64-1.80 (m, 2H, —C—CH₂—C—),3.10-3.31 (m, 4H, piperazine ring), 3.54 (d, 1H, J=1.7 Hz, epoxy ring),3.58-3.93 (m, 4H, piperazine ring), 3.62 (d, 1H, J=1.7 Hz, epoxy ring),5.04-5.12 (m, 1H, —N—CH—CO), 4.8-6.5 (brd, 1H, —COOH), 6.82-6.88 (m, 2H,aromatic), 7.21-7.26 (m, 1H, aromatic), 8.18 (d, 1H, J=8.9 Hz, —NH—).

Example 33

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-methoxyphenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(4-methoxyphenyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]-oxiranecarboxylicacid (0.49 g, 56.7%; Compound 20).

¹H NMR (CDCl₃) δ: 0.95 (d, 3H, J=6.3 Hz, —C—CH₃), 0.99 (d, 3H, J=6.6 Hz,—C—CH₃), 1.38-1.46 (m, 1H, —C—CH—C₂), 1.63-1.80 (m, 2H, —C—CH₂—C—),3.05-3.19 (m, 4H, piperazine ring), 3.55 (d, 1H, J=1.7 Hz, epoxy ring),3.60-3.90 (m, 4H, piperazine ring), 3.62 (d, 1H, J=1.7 Hz, epoxy ring),5.06-5.13 (m, 1H, —N—CH—CO), 4.8-5.8 (brd, 1H, —COOH), 6.84-7.00, (m,4H, aromatic), 8.14 (d, 1H, J=8.6 Hz, —NH—).

Example 34

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-methyl]propyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-methyl]propyl]amino]-carbonyl]oxiranecarboxylicacid (1.09 g, 89.5%; Compound 21) as colorless crystals.

¹H NMR (CDCl₃) δ: 0.98 (d,3H, J=6.6 Hz, —C—CH₃), 1.01 (d, 3H, J=6.6 Hz,—C—CH₃, 2.10 (m, 1H, —CH—C₂), 2.98-3.14 (m, 4H, piperazine ring), 3.64(d, J=1.6 Hz, 1H, epoxy ring), 3.66 (d, 1H, J=1.6 Hz, epoxy ring),3.68-4.01 (m, 4H, piperazine ring), 4.98 (dd, 1H, J=8.9, 5.9 Hz,—N—CH—CO), 7.00-7.06 (m, 2H, aromatic), 7.24 (m, 1H, aromatic), 7.39 (m,1H, aromatic), 8.29 (d, 1H, J=8.9, —NH).

Example 35

Using ethyl(2s,3s)-3-[[[[[4-(2-chlorophenyl)-1-piperazinyl]-carbonyl]methyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]methyl]amino]carbonyl]oxiranecarboxylicacid (1.08 g, 66.5%; Compound 22) as colorless crystals.

¹H NMR (CDCl₃) δ: 3.02-3.09 (m, 4H, piperazine ring), 3.63 (m, 2H,piperazine ring), 3.66 (d, 1H, J=1.6 Hz, epoxy ring), 3.78 (d, 1H, J=1.6Hz, epoxy ring), 3.80 (m, 2H, piperazine ring), 4.11 (dd, 1H, J=17.0,5.4 Hz, —N—CH—CO), 4.33 (dd, 1H, J=17.0, 5.4 Hz, —N—CH—CO), 6.99-7.05(m, 2H, aromatic), 7.23 (m, 1H, aromatic), 7.38 (m, 1H, aromatic), 8.67(brd, 1H, —NH).

Example 36

Using ethyl(2s,3s)-3-[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]ethyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]ethyl]amino]carbonyl]oxiranecarboxylicacid (0.87 g, 77.5%; Compound 23) as colorless crystals.

¹H NMR (CDCl₃) δ: 1.41 (d, 3H, J=6.8 Hz, —C—CH3), 2.98-3.13 (m, 4H,piperazine ring), 3.61 (d, 1H, J=1.6 Hz, epoxy ring), 3.63 (d, 1H, J=1.6Hz, epoxy ring), 3.67-3.84 (m, 3H, piperazine ring), 3.95 (m, 1H,piperazine ring), 5.06 (dq, 1H, J=8.4, 6.8 Hz, —N—CH—CO), 7.00-7.06 (m,2H, aromatic), 7.24 (m, 1H, aromatic), 7.39 (m, 1H, aromatic), 8.13 (d,1H, J=8.4, —NH).

Example 37

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-methyl]butyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-methyl]butyl]amino]carbonyl]-oxiranecarboxylicacid (1.07 g, 77.4%; Compound 24) as colorless crystals.

¹H NMR (CDCl₃) δ: 0.92 (t, 3H, J=7.3 Hz, —C—CH₃), 1.00 (d, 3H, J=6.8 Hz,—C—CH₃), 1.22 (m, 1H, —CH—C₂—), 1.54 (m, 1H, —CH—C), 1.84 (m, 1H,—CH—C), 2.97-3.17 (m, 4H, piperazine ring), 3.60 (d, 1H, J=1.6 Hz, epoxyring), 3.64 (d, 1H, J=1.6 Hz, epoxy ring), 3.65-3.79 (m, 2H, piperazinering), 3.85-4.05 (m, 2H, piperazine ring), 4.98 (dd, 1H, J=9.2, 6.3 Hz,—N—CH—CO), 7.00-7.06 (m, 2H, aromatic), 7.24 (m, 1H, aromatic), 7.39 (m,1H, aromatic), 8.29 (d, 1H, J=9.2, —NH).

Example 38

Using ethyl(2s,3s)-3-[[[2-[[4-(2-chlorophenyl)-1-piperazinyl]-carbonyl]ethyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[2-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]ethyl]amino]carbonyl]oxiranecarboxylicacid (0.85 g, 78.9%; Compound 25) as colorless crystals.

¹H NMR DMSO-d₆) δ: 2.56 (t, 2H, J=6.9 Hz, —C—CH₂—CO), 2.91-2.98 (m, 4H,piperazine ring), 3.35 (td, 2H, J=6.9, 5.6 Hz, N—CH₂—C), 3.49 (d, 1H,J=2.0 Hz, epoxy ring), 3.59 (d, 1H, J=1.6 Hz, epoxy ring), 3.56-3.64 (m,2H, piperazine ring), 3.80 (m, 2H, piperazine ring), 7.07 (td, 1H,J=7.9, 1.7 Hz, aromatic), 7.15 (dd, 1H, J=7.9, 1.7 Hz, aromatic), 7.43(dd, 1H, J=7.9, 1.7 Hz, aromatic), 7.31 (m, 1H, aromatic), 8.40 (t, 1H,J=5.6 Hz, —NH), 13.50 (brd, 1H, —COOH).

Example 39

Using ethyl(2s,3s)-3-[[[[N-[[4-(2-chlorophenyl)-1-piperazinyl]-carbonyl]methyl]-N-methyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[N-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]methyl]-N-methyl]amino]carbonyl]oxiranecarboxylicacid (1.08 g, 74.8%; Compound 26) as colorless crystals.

¹H NMR (CDCl₃) δ: 3.01-3.10 (m, 6H, piperazine ring), 3.27 (s, 3H,—NCH₃), 3.63-3.71 (m, 2H, —N—CH₂—CO), 3.75 (d, 1H, J=1.9 Hz, epoxyring), 3.78-3.90 (m, 2H, piperazine ring), 4.02 (d, 1H, J=1.9 Hz, epoxyring), 6.98-7.05 (m, 2H, aromatic), 7.24 (m, 1H, aromatic), 7.36 (m, 1H,aromatic).

Example 40

Using ethyl(2s,3s)-3-[[(2s)-2-[[4-(2-chlorophenyl)-1-piperazinyl]-carbonyl]-1-pyrrolidinyl]carbonyl]-oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[(2s)-2-[[4-(2-chloro-phenyl)-1-piperazinyl]carbonyl]-1-pyrrolidinyl]carbonyl]oxiranecarboxylicacid (0.94 g, 7.07%; Compound 27) as colorless crystals.

¹H NMR (CDCl₃) δ: 1.94-2.11 (m, 2H, pyrrolidine ring), 2.17-2.30 (m, 2H,pyrrolidine ring), 3.06-3.20 (m, 4H, piperazine ring), 3.63-3.76 (m, 2H,piperazine ring), 3.81-3.85 (m, 5H), 4.00 (dt, 1H, J=13.7, 4.4 Hz,pyrrolidine ring), 4.96 (dd, 1H, J=7.8, 4.3 Hz, pyrrolidine ring),6.97-7.04 (m, 2H, aromatic), 7.24 (m, 1H, aromatic), 7.37 (m, 1H,aromatic).

Example 41

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-acetylaminomethylthio]ethyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-2-acetylaminomethylthio]-ethyl]amino]carbonyl]oxiranecarboxylicacid (0.88 g, 77.9%; Compound 28) as colorless crystals.

¹H NMR (CDCl₃) δ: 2.05 (s, 3H, —COCH₃), 2.85 (dd, 1H, J=13.9, 8.3 Hz,—C—CH—S), 2.96-3.16 (m, 5H), 3.69 (d, 1H, J=1.6 Hz, epoxy ring), 3.78(d, 1H, J=1.6 Hz, epoxy ring), 3.71-3.89 (m, 4H, piperazine ring), 4.39(d, 2H, J=8.3 Hz, —S—CH₂—N), 5.21 (m, 1H, —N—CH—CO), 6.97-7.02 (m, 2H,aromatic), 7.22 (m, 1H, aromatic), 7.36 (m, 1H, aromatic), 7.80 (d, 1H,J=8.3, —NH), 9.00 (brd, 1H, —NH).

Example 42

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-3-methylthio]propyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-3-methylthio]propyl]amino]-carbonyl]oxiranecarboxylicacid (1.17 g, 80.7%; Compound 29) as colorless crystals.

¹H NMR (CDCl₃) δ: 1.98 (dd, 2H, J=6.9, 6.6 Hz, —CH—C—C), 2.12 (s, 3H,—SCH₃), 2.57 (dt, 2H, J=6.9, 2.3 Hz, —C—CH₂—C—S), 3.05-3.19 (m, 4H,piperazine ring), 3.63 (d, 1H, J=1.9 Hz, epoxy ring), 3.65 (d, 1H, J=1.9Hz, epoxy ring), 3.71-3.94 (m, 4H, piperazine ring), 5.26 (m, 1H,—N—CH—CO), 7.00-7.06 (m, 2H, aromatic), 7.24 (m, 1H, aromatic), 7.38 (m,1H, aromatic), 8.18 (d, 1H, J=8.6, —NH).

Example 43

Using ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-3-carbamoyl]propyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[[(1s)-1-[[4-(2-chlorophenyl)-1-piperazinyl]carbonyl]-3-carbamoyl]propyl]amino]-carbonyl]oxiranecarboxylicacid (0.14 g, 74.5%; Compound 30) as colorless crystals.

¹H NMR (CDCl₃) δ: 1.85 (brd, 2H, —NH2), 2.14 (m, 1H, —C—CH—C—CO—),2.36-2.53 (m, 3H, —CH—CH₂—C—CO—), 2.89-3.06 (m, 4H, piperazine ring),3.57-3.79 (m, 6H, piperazine and epoxy ring), 5.02 (m, 1H, —N—CH—CO),6.96-7.02 (m, 2H, aromatic), 7.21 (m, 1H, aromatic), 7.37 (m, 1H,aromatic), 7.88 (brd, 1H, —NH).

Example 44

Using ethyl(2s,3s)-3-[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-1-cyclopentyl]amino]carbonyl]oxiranecarboxylateinstead of ethyl(2s,3s)-3-[[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]-carbonyl]-2-phenyl]ethyl]amino]carbonyl]oxiranecarboxylate,the procedure of Example 24 was repeated to give(2s,3s)-3-[[[(1s)-1-[[4-(4-fluorophenyl)-1-piperazinyl]carbonyl]-1-cyclopentyl]amino]carbonyl]-oxiranecarboxylicacid (0.52 g, 55.6%; Compound 31) as colorless crystals.

¹H NMR DMSO-d₆) δ: 1.61 (m, 4H, cyclopentyl), 1.87 (m, 2H, cyclopentyl),2.22 (m, 2H, cyclopentyl), 2.97 (m, 4H, piperazine), 3.45 (d, 1H, J=1.6Hz, epoxy ring), 3.58 (d, 1H, J=2.1 Hz, epoxy ring), 3.60 (m, 4H,piperazine ring), 6.95-7.20 (m, 4H, aromatic), 8.89 (s, 1H, —NH), 13.4(brd, 1H, —COOH).

Example 45

To a solution of the(2s,3s)-3-[[[[(1s)-1-[[4-(4-methylphenyl-sulfonyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]-oxiranecarboxylicacid (0.935 g, 2 mmol) obtained in Example 29 in dichloromethane (15 ml)were added o-benzylhydroxylamine (0.638 g, 4.0 mmol) andN-methylmorpholine (0.405 g, 4.0 mmol). Then, a solution ofdicyclohexylcarbodiimide (0.619 g, 3.0 mmol) in dichloromethane (5 ml)was added dropwise under ice-cooling. The mixture was stirred at roomtemperature for 24 hours, at the end of which time it was filtered. Theprecipitate was washed with dichloromethane (20 ml) and the washing andthe filtrate were pooled and washed with water. The organic layer wasdried over anhydrous magnesium sulfate and concentrated under reducedpressure and the residue was chromatographed on silica gel. Elution wascarried out with ethyl acetate-hexane (2:1, v/v) to provide(2s,3s)-3-[[[[(1s)-1-[[4-(4-methylphenylsulfonyl)-1-piperazinyl]-carbonyl]-3-methyl]butyl]amino]carbonyl]oxiranecarbobenzyloxamide(0.86 g, 75.1%; Compound 32).

¹H NMR (CDCl₃) δ: 0.84 (d, 3H, J=6.2 Hz, —C—CH₃), 0.91 (d, 3H, J=6.5 Hz,—C—CH₃), 1.23-1.31 (m, 1H, —C—CH₂—C), 1.36-1.58 (m, 2H, —C—CH—C,—C—CH—C₂), 2.43 (s, 3H, -ph-CH₃), 2.72-2.86 (m, 2H, piperazine ring),3.14-3.27 (m, 2H, piperazine ring), 3.31-3.51 (m, 2H, piperazine ring),3.40 (d, 1H, J=1.4 Hz, epoxy ring), 3.43 (d, 1H, J=1.7 Hz, epoxy ring),3.63-3.74 (m, 1H, piperazine ring), 3.84-3.98 (m, 1H, piperazine ring),4.80-4.90 (m, 1H, —N—CH—CO), 4.87 (s, 3H, —O—CH2-ph), 7.30-7.40 (m, 8H,aromatic, —NH—), 7.56-7.66 (m, 2H, aromatic), 9.05 (s, 1H, —NH—).

Example 46

To a solution of the(2s,3s)-3-[[[[(1s)-1-[[4-(4-methylphenyl-sulfonyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]-oxiranecarbobenzyloxamide(0.57 g, 1 mmol) obtained in Example 45 in methanol (25 ml) was added acatalyst amount of palladium-on-carbon and catalytic reduction wascarried out. After completion of the reaction, the palladium-on-carbonwas filtered off and the filtrate was concentrated and chromatographedon silica gel. Elution was carried out with ethyl acetate to provide(2s,3s)-3-[[[[(1s)-1-[[4-(4-methylphenyl-sulfonyl)-1-piperazinyl]carbonyl]-3-methyl]butyl]amino]carbonyl]-oxiranecarbohydroxamicacid (0.18 g, 37.3%; Compound 33).

¹H NMR (CDCl₃) δ: 0.84 (d, 3H, J=5.9 Hz, —C—CH₃), 0.90 (d, 3H, J=5.9 Hz,—C—CH₃), 1.24-1.33 (m, 1H, —C—CH₂—C), 1.50-1.64 (m, 2H, —C—CH—C,—C—CH—C₂), 2.42 (s, 3H, -ph-CH₃), 2.90-3.20 (m, 4H, piperazine ring),3.44-3.80 (m, 3H, piperazine ring), 3.51 (s, 1H, epoxy ring), 3.68 (s,1H, epoxy ring), 4.56-4.66 (m, 1H, piperazine ring), 4.76-4.90 (m, 1H,—N—CH—CO), 7.33 (d, 2H, J=7.8 Hz, aromatic), 7.62 (dd, 2H, J=7.8, 1.7Hz, aromatic), 7.84-7.94 (brd, 1H, —NH—), 9.80-10.40 (brd, 1H, —OH).

Example 47 Tablets

Compound 23 80 mg Starch 17 mg Magnesium stearate  3 mg

The above components per tablet are compressed into tablets in theroutine manner. Where necessary, the tablets can be sugar-coated.

Example 48 Capsules

Compound 18 50 mg Lactose 100 mg  Starch 30 mg Magnesium stearate 10 mg

The above components per tablet are mixed and filled in gelatin capsuleshells.

Example 49 Injection

Compound 21  2.5 mg Sodium chloride 900 mg 1N-sodium hydroxide q.s.Distilled water for injection to make 100 ml 

The above components are mixed in the routine manner to provide aninjection.

Example 50 Ophthalmic solution

Compound 18 50 mg Boric acid 700 mg Borax q.s. Sodium chloride 500 mgSodium edetate 0.05 mg Benzalkonium chloride 0.005 mg Sterilized purewater to make 100 ml

The above components are mixed in the routine manner to provide anophthalmic solution.

Example 51 N-(2-Naphthalenesulfonyl)-L-valyl-L-leucinal

Valine (11.5 g) was dissolved in 1M aqueous sodium hydroxide solution(100 ml), and purified water (200 ml) and tetrahydrofuran (100 ml) wereadded. Thereto were simultaneously added dropwise 1M aqueous sodiumhydroxide solution (100 ml) and a solutiion (100 ml) of2-naphthalenesulfonyl chloride (18.5 g) in tetrahydrofuran with stirringunder ice-cooling. The solution was stirred for one day at roomtemperature to allow reaction. After the completion of the reaction, thereaction mixture was adjusted to pH 2-3 and extracted with ethylacetate. The extract was washed with dilute hydrochloric acid andsaturated brine, and dried over anhydrous magnesium sulfate. Ethylacetate was evaporated under reduced pressure, and the residue waswashed with a mixture of hexane-ethyl acetate to give 12.8 g ofN-(2-naphthalenesulfonyl)-L-valine as white crystals.

N-(2-Naphthalenesulfonyl)-L-valine (12. 0 g) and N-hydroxysuccinimide(5.4 g) were dissolved in tetrahydrofuran (200 ml), and a solution (200ml) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (9.0g) in dichloromethane was gradually added dropwise with stirring underice-cooling. The solution was stirred for 4 hr at room temperature toallow reaction. After the completion of the reaction, the solvent wasevaporated under reduced pressure and the residue was dissolved in ethylacetate. The mixture was washed with dilute hydrochloric acid, saturatedaqueous solution of sodium hydrogen-carbonate and saturated brine, anddried over anhydrous magnesium sulfate. Ethyl acetate was evaporatedunder reduced pressure, and the residue was washed with a mixed solutionof hexane-ethyl acetate to give 14.1 g ofN-(2-naphthalenesulfonyl)-L-valine N-hydroxysuccinimide ester as whitecrystals.

N-(2-Naphthalenesulfonyl)-L-valine N-hydroxysuccinimide ester (1.8 g)and leucinol (0.63 g) were added to dichloromethane (100 ml), and themixture was stirred at room temperature while adding triethylamine (0.68g). The solution was stirred for 2 hr to allow reaction. After thecompletion of the reaction, the mixture was washed with dilutehydrochloric acid, saturated aqueous solution of sodiumhydrogen-carbonate and saturated brine, and dried over anhydrousmagnesium sulfate. Dichloromethane was evaporated under reducedpressure, and the residue was washed with a mixed solution ofhexane-ethyl acetate to give 1.3 g ofN-(2-naphthalenesulfonyl)-L-valyl-L-leucinol as white crystals.

N-(2-Naphthalenesulfonyl)-L-valyl-L-leucinol (1.3 g) was dissolved indimethyl sulfoxide (20 ml) and dichloromethane (10 ml), andtriethylamine (1.9 g) was added. The solution was stirred at roomtemperature while adding a solution (20 ml) of sulfur trioxide-pyridinecomplex (2.0 g) in dimethyl sulfoxide, which was followed by stirringfor 2 hr. After the completion of the reaction, ethyl acetate was added.The mixture was washed with dilute hydrochloric acid, saturated aqueoussolution of sodium hydrogencarbonate and saturated brine, and dried overanhydrous magnesium sulfate. The solvent was evaporated under reducedpressure, and the residue was washed with a mixed solution ofhexane-ethyl acetate to give 0.98 g ofN-(2-naphthalenesulfonyl)-L-valyl-L-leucinal (Compound 34) as whitecrystals. [Step 1]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.42 (d, 3H, J=6.3 Hz), 0.55 (d, 3H, J=6.3Hz), 0.84 (d, 3H, J=6.6 Hz), 0.88 (d, 3H, J=6.6 Hz), 0.93-1.12 (m, 2H),1.14-1.28 (m, 1H), 1.82-2.00 (m, 1H), 3.63-3.72 (m, 2H), 7.62-8.40 (m,9H), 9.02 (s, 1H).

Anal. (C₂₁H₂₈N₂O₄S) C, H, N.

In the same manner as in Step 1 except that 4-fluorobenzenesulfonylchloride was used instead of 2-naphthalenesulfonyl chloride of Step 1,N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (Compound 35) was obtainedas white crystals. [Step 2]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.74 (d, 3H, J=5.9 Hz), 0.80 (d, 6H, J=6.4Hz), 0.85 (d, 3H, J=6.8 Hz), 1.14-1.46 (m, 3H), 1.81-1.93 (m, 1H),3.56-3.62 (dd, 1H, J=6.6, 9.5 Hz), 3.80-3.88 (m, 1H), 7.33-7.42 (m, 2H),7.79-7.86 (m, 2H), 7.96 (d, 1H, J=9.8 Hz), 8.27 (d, 1H, J=7.3 Hz), 9.14(s, 1H).

Anal. (C₁₇H₂₅FN₂O₄S) C, H, N.

In the same manner as in Step 1 except that 4-chlorobenzenesulfonylchloride was used instead of 2-naphthalenesulfonyl chloride of Step 1,N-(4-chlorophenylsulfonyl)-L-valyl-L-leucinal (Compound 36) was obtainedas white crystals. [Step 3]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.74 (d, 3H, J=5.9 Hz), 0.82 (d, 6H, J=6.8Hz), 0.88 (d, 3H, J=6.3 Hz), 1.15-1.46 (m, 1H), 3.61 (dd, 1H, J=6.8, 9.3Hz), 3.82-3.90 (m, 1H), 7.56-7.63 (m, 2H), 7.44-7.79 (m, 2H), 8.03 (d,1H, J=9.3 Hz), 8.26 (d, 1H, J=7.3 Hz), 9.15 (s, 1H).

Anal. (C₁₇H₂₅ClN₂O₄S) C, H, N.

In the same manner as in Step 1 except that p-toluenesulfonyl chloridewas used instead of 2-naphthalenesulfonyl chloride of Step 1,N-(4-methylphenylsulfonyl)-L-valyl-L-leucinal (Compound 37) was obtainedas white crystals. [Step 4]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.72-0.90 (m, 12H), 1.18-1.45 (m, 3H),1.79-1.91 (m, 1H), 2.36 (s, 3H), 3.57 (t, 1H, J=7.7 Hz), 3.77-3.84 (m,1H), 7.32 (d, 2H), 7.62-7.70 (m, 2H), 7.76 (d, 1H, J=8.3 Hz), 8.26 (d,1H, J=6.8 Hz), 9.07 (s, 1H).

Anal. (C₁₈H₂₈N₂O₄S) C, H, N.

In the same manner as in Step 1 except that tert-leucine was usedinstead of valine of Step 1,N-(2-naphthalenesulfonyl)-L-tert-leucyl-L-leucinal (Compound 38) wasobtained as white crystals. [Step 5]

¹H-NMR DMSO-d₆ 270 MHz) δ: 0.35 (d, 3H, J=6.4 Hz), 0.46 (d, 3H, J=6.4Hz), 0.78-0.95 (m, 2H), 0.95 (s, 9H), 1.08-1.20 (m, 1H), 3.45-3.55 (m,1H), 3.67 (d, 1H, J=10.3 Hz), 7.62-7.72 (m, 2H), 7.82-7.86 (m, 1H),7.97-8.10 (m, 4H), 8.17 (d, 1H, J=6.4 Hz), 8.29 (m, 1H), 8.91 (s, 1H).

Anal. (C₂₂H₃₀N₂O₄S) C, H, N.

In the same manner as in Step 1 except that 4-fluorobenzenesulfonylchloride was used instead of 2-naphthalenesulfonyl chloride, andD-valine was used instead of valine of Step 1,N-(4-fluorophenylsulfonyl)-D-valyl-L-leucinal (Compound 39) was obtainedas white crystals. [Step 6]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.78 (d, 3H, J=6.3 Hz), 0.82 (d, 3H, J=6.9Hz), 0.83 (d, 6H, J=6.3 Hz), 1.24-1.50 (m, 3H), 1.80-1.92 (m, 1H), 3.62(s br, 1H), 3.84-3.92 (m, 1H), 7.32-7.41 (m, 2H), 7.79 (m, 3H), 8.33 (d,1H, J=6.9 Hz), 8.96 (s, 1H).

Anal. (C₂₂H₃₀N₂O₄S) C, H, N.

In the same manner as in Step 1 except that 4-fluorobenzenesulfonylchloride was used instead of 2-naphthalenesulfonyl chloride, andnorleucine was used instead of valine of Step 1,N-(4-fluorophenyl-sulfonyl)-L-norleucyl-L-leucinal (Compound 40) wasobtained as white crystals. [Step 7]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.74-0.90 (m, 9H), 1.07-1.59 (m, 9H), 3.76(t, 1H, J=5.4 Hz), 3.84-3.91 (m, 1H), 7.34-7.45 (m, 2H), 7.79-8.07 (m,3H), 8.29 (d, 1H, J=7.3 Hz), 9.18 (s, 1H).

Anal. (C₂₂H₃₀N₂O₄S) C, H, N.

In the same manner as in Step 1 except that 4-fluorobenzenesulfonylchloride was used instead of 2-naphthalenesulfonyl chloride, andnorvaline was used instead of valine of Step 1,N-(4-fluorophenyl-sulfonyl)-L-norvalyl-L-leucinal (Compound 41) wasobtained as white crystals. [Step 8]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.69-0.85 (m, 9H), 1.14-1.66 (m, 7H), 3.78(t, 1H, J=6.3 Hz), 3.84-3.92 (m, 1H), 7.34-7.42 (m, 2H), 7.79-8.02 (m,3H), 8.28 (d, 1H, J=7.3 Hz), 9.18 (s, 1H).

Anal. (C₂₂H₃₀N₂O₄S) C, H, N.

Example 52 N-(2-Naphthalenesulfonyl)-L-tert-leucyl-L-phenylalaninal

tert-Leucine (13.1 g) was dissolved in 1M aqueous sodium hydroxidesolution (100 ml), and purified water (200 ml) and tetrahydrofuran (100ml) were added. Thereto were simultaneously added dropwise 1M aqueoussodium hydroxide solution (100 ml) and a solution (100 ml) of2-naphthalenesulfonyl chloride (20.4 g) in tetrahydrofuran with stirringunder ice-cooling. The solution was stirred for one day at roomtemperature to allow reaction. After the completion of the reaction, thereaction mixture was adjusted to pH 2-3 and extracted with ethylacetate. The extract was washed with dilute hydrochloric acid andsaturated brine, and dried over anhydrous magnesium sulfate. Ethylacetate was evaporated under reduced pressure, and the residue waswashed with a mixture of hexane-ethyl acetate to give 16.5 g ofN-(2-naphthalenesulfonyl)-L-tert-leucine as white crystals.

N-(2-Naphthalenesulfonyl)-L-tert-leucine (16.0 g) andN-hydroxysuccinimide (6.9 g) were dissolved in tetrahydrofuran (200 ml),and a solution (200 ml) of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (11.5 g) indichloromethane was gradually added dropwise with stirring underice-cooling. The solution was stirred at room temperature for about 12hr to allow reaction. After the completion of the reaction, the solventwas evaporated under reduced pressure and the residue was dissolved inethyl acetate. The mixture was washed with dilute hydrochloric acid,saturated aqueous solution of sodium hydrogencarbonate and saturatedbrine, and dried over anhydrous magnesium sulfate. Ethyl acetate wasevaporated under reduced pressure, and the residue was washed with amixed solution of hexane-ethyl acetate to give 18.3 g ofN-(2-naphthalenesulfonyl)-L-tert-leucine N-hydroxysuccinimide ester aswhite crystals.

N-(2-Naphthalenesulfonyl)-L-tert-leucine N-hydroxysuccinimide ester (1.8g) and phenylalaninol (1.0 g) were added to dichloromethane (50 ml), andthe mixture was stirred at room temperature while adding triethylamine(0.86 g). The solution was stirred for 2 hr to allow reaction. After thecompletion of the reaction, the mixture was washed with dilutehydrochloric acid, saturated aqueous solution of sodiumhydrogencarbonate and saturated brine, and dried over anhydrousmagnesium sulfate. Dichloromethane was evaporated under reducedpressure, and the residue was washed with a mixed solution ofhexane-ethyl acetate to give 1.6 g ofN-(2-naphthalenesulfonyl)-L-tert-leucyl-L-phenylalaninol as whitecrystals.

N-(2-Naphthalenesulfonyl)-L-tert-leucyl-L-phenylalaninol (1.6 g) wasdissolved in dimethyl sulfoxide (20 ml) and dichloromethane (10 ml), andtriethylamine (2.1 g) was added. The solution was stirred at roomtemperature while adding a solution (15 ml) of sulfur trioxide-pyridinecomplex (2.2 g) in dimethyl sulfoxide, which was followed by stirringfor 2 hr. After the completion of the reaction, ethyl acetate was added.The mixture was washed with dilute hydrochloric acid, saturated aqueoussolution of sodium hydrogencarbonate and saturated brine, and dried overanhydrous magnesium sulfate. The solvent was evaporated under reducedpressure, and the residue was washed with a mixed solution ofhexane-ethyl acetate to give 1.1 g ofN-(2-naphthalene-sulfonyl)-L-tert-leucyl-L-phenylalaninal (Compound 42)as white crystals. [Step 1]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.86 (s, 9H), 2.26-2.40 (m, 1H), 2.63-2.77(m, 1H), 3.56 (dd, 1H, J=6.8, 13.2 Hz), 3.63-3.68 (m, 1H), 6.87-6.90 (m,1H), 6.99-7.03 (m, 1H), 7.11-7.22 (m, 3H), 7.60-7.72 (m, 2H), 7.80-7.87(m, 1H), 7.92-8.19 (m, 4H), 8.35 (d, 1H, J=6.8 Hz), 8.40-8.43 (m, 1H),8.63 (s, 1H).

Anal. (C₂₅H₂₈N₂O₄S) C, H, N.

In the same manner as in Step 1 except that 4-fluorobenzenesulfonylchloride was used instead of 2-naphthalenesulfonyl chloride, and valinewas used instead of tert-leucine of Step 1,N-(4-fluorophenylsulfonyl)-L-valyl-L-phenylalaninal (Compound 43) wasobtained as white crystals. [Step 2]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.76 (d, 3H, J=6.4 Hz), 0.77 (d, 3H, J=6.4Hz), 1.69-1.86 (m, 1H), 2.67 (dd, 1H, J=8.8, 14.2 Hz), 3.02 (dd, 1H,J=5.1, 14.2 Hz), 3.56 (dd, 1H, J=6.4, 9.3 Hz), 3.99-4.07 (m, 1H),7.12-7.29 (m, 7H), 7.72-7.84 (m, 2H), 7.92 (d, 1H, J=9.3 Hz), 8.44 (d,1H, J=6.8 Hz), 9.07 (s, 1H).

Anal. (C₂₀H₂₃FN₂O₄S) C, H, N.

In the same manner as in Step 1 except that valine was used instead oftert-leucine of Step 1,N-(2-naphthalenesulfonyl)-L-valyl-L-phenylalaninal (Compound 44) wasobtained as white crystals. [Step 3]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.63 (d, 3H, J=6.6 Hz), 0.76 (d, 3H, J=6.6Hz), 1.68-1.82 (m, 1H,), 2.40-2.92 (m, 1H), 3.64 (dd, 1H, J=6.6, 9.2Hz), 3.97-3.87 (m, 1H), 6.95-7.02 (m, 2H), 7.10-7.23 (m, 3H), 7.62-7.82(m, 3H), 7.94-8.10 (m, 4H), 8.36-8.43 (m, 2H), 8.86 (s, 1H).

Anal. (C₂₄H₂₆N₂O₄S) C, H, N.

In the same manner as in Step 1 except that 4-chlorobenzenesulfonylchloride was used instead of 2-naphthalenesulfonyl chloride, and valinewas used instead of tert-leucine of Step 1,N-(4-chlorophenylsulfonyl)-L-valyl-L-phenylalaninal (Compound 45) wasobtained as white crystals. [Step 4]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.77 (d, 3H, J=6.8 Hz), 0.79 (d, 3H, J=6.8Hz), 1.70-1.87 (m, 1H), 2.67 (dd, 1H, J=8.8, 14.2 Hz), 3.01 (dd, 1H,J=5.4, 14.2 Hz), 3.60 (dd, 1H, J=6.4, 9.3 Hz), 4.00-4.07 (m, 1H),7.12-7.32 (m, 5H), 7.50-7.60 (m, 2H), 7.68-8.00 (m, 2H), 7.98 (d, 1H,J=9.3 Hz), 8.44 (d, 1H, J=6.8 Hz), 9.09 (s, 1H).

Anal. (C₂₀H₂₃ClN₂O₄S) C, H, N.

In the same manner as in Step 1 except that p-toluenesulfonyl chloridewas used instead of 2-naphthalenesulfonyl chloride, and valine was usedinstead of tert-leucine of Step 1,N-(4-methylphenylsulfonyl)-L-valyl-L-phenylalaninal (Compound 46) wasobtained as white crystals. [Step 5]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.74 (d, 6H, J=6.4 Hz), 1.71-1.81 (m, 1H),2.33 (s, 3H), 2.65 (dd, 1H, J=8.8, 14.2), 2.99 (dd, 1H, J=5.4, 14.2),3.55 (dd, 1H, J=6.4, 9.3 Hz), 3.97-4.05 (m, 1H), 7.11-7.37 (m, 7H),7.59-7.66 (m, 2H), 7.73 (d, 1H, J=9.3 Hz), 8.41 (d, 1H, J=6.8 Hz), 8.99(s, 1H).

Anal. (C₂₁H₂₆N₂O₄S) C, H, N.

In the same manner as in Step 1 except that1-aminocyclohexane-carboxylic acid was used instead of tert-leucine ofStep 1,1-(2-naphthalenesulfonylamino)cyclohexanecarbonyl-L-phenylalaninol(Compound 47) was obtained as white crystals. [Step 6]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 112 (s br, 6H), 1.65 (s br, 4H), 2.28 (dd,1H, J=8.6, 14.2 Hz), 3.06 (dd, 1H, J=5.3, 14.2 Hz), 4.07-4.14 (m, 1H),7.16-7.29 (m, 5H), 7.63-7.72 (m, 2H), 7.86-7.72 (m, 2H), 7.98-8.15 (m,4H), 8.41 (s, 1H), 9.29 (s, 1H).

Anal. (C₂₆H₂₈N₂O₄S) C, H, N.

Example 53 N-(4-Chlorophenylsulfonyl)-L-valyl-L-tryptophanal

Valine (13.1 g) was dissolved in 1M aqueous sodium hydroxide solution(100 ml), and purified water (250 ml) and tetrahydrofuran (100 ml) wereadded. Thereto were alternately added 1M aqueous sodium hydroxidesolution (100 ml) and a solution (100 ml) of 4-chlorobenzenesulfonylchloride (19.0 g) in tetrahydrofuran in ⅕ portions thereof with stirringunder ice-cooling. The solution was stirred for one day at roomtemperature to allow reaction. After the completion of the reaction, thereaction mixture was adjusted to pH 2-3 and extracted with ethylacetate. The extract was washed with dilute hydrochloric acid andsaturated brine, and dried over anhydrous magnesium sulfate. Ethylacetate was evaporated under reduced pressure, and the residue waswashed with a mixture of hexane-ethyl acetate to give 13.6 g ofN-(4-chlorophenylsulfonyl)-L-valine as white crystals.

N-(4-Chlorophenylsulfonyl)-L-valine (13.5 g) and N-hydroxysuccinimide(6.4 g) were dissolved in tetrahydrofuran (200 ml), and a solution (200ml) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (10.6g) in dichloromethane was gradually added dropwise with stirring underice-cooling. The solution was stirred at room temperature for about 12hr to allow reaction. After the completion of the reaction, the solventwas evaporated under reduced pressure and the residue was dissolved inethyl acetate. The mixture was washed with dilute hydrochloric acid,saturated aqueous solution of sodium hydrogencarbonate and saturatedbrine, and dried over anhydrous magnesium sulfate. Ethyl acetate wasevaporated under reduced pressure, and the residue was washed with amixed solution of hexane-ethyl acetate to give 14.3 g ofN-(4-chlorophenylsulfonyl)-L-valine N-hydroxysuccinimide ester as whitecrystals.

N-(4-Chlorophenylsulfonyl)-L-valine N-hydroxysuccinimide ester (1.5 g)and L-tryptophanol (0.88 g) were added to dichioromethane (100 ml), andthe mixture was stirred at room temperature while adding triethylamine(1.2 g). The solution was stirred for 2 hr to allow reaction. After thecompletion of the reaction, the solvent was evaporated under reducedpressure. The residue was dissolved in ethyl acetate, washed with dilutehydrochloric acid, saturated aqueous solution of sodiumhydrogencarbonate and saturated brine, and dried over anhydrousmagnesium sulfate. Ethyl acetate was evaporated under reduced pressure,and the residue was washed with a mixed solution of hexane-ethyl acetateto give 1.6 g of N-(4-chlorophenylsulfonyl)-L-valyl-L-triptophanol aswhite crystals.

N-(4-Chlorophenylsulfonyl)-L-valyl-L-tryptophanol (1.5 g) was dissolvedin dimethyl sulfoxide (20 ml) and dichloromethane (15 ml), andtriethylamine (2.0 g) was added. The solution was stirred at roomtemperature while adding a solution (20 ml) of sulfur trioxide-pyridinecomplex (2.1 g) in dimethyl sulfoxide, which was followed by stirringfor 1 hr. After the completion of the reaction, ethyl acetate was added.The mixture was washed with dilute hydrochloric acid, saturated aqueoussolution of sodium hydrogencarbonate and saturated brine, and dried overanhydrous magnesium sulfate. The solvent was evaporated under reducedpressure, and the residue was purified by preparative TLC plate(developing solvent: hexane-ethyl acetate, 1:1, v/v) to give 0.10 g ofN-(4-chlorophenylsulfonyl)-L-valyl-L-tryptophanal (Compound 48) as whitecrystals. [Step 1]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.81 (d, 3H, J=6.8 Hz), 0.82 (d, 3H, J=6.4Hz), 1.77-1.91(m, 1H), 2.82 (dd, 1H, J=7.8, 15.1 Hz), 3.07 (dd, 1H,J=5.9, 15.1 Hz), 3.65 (dd, 1H, J=6.8, 9.3 Hz), 4.06-4.14 (m, 1H),6.96-7.69 (m, 9H), 7.99 (d, 1H, J=9.8 Hz), 8.41 (d, 1H, J=6.4 Hz), 9.21(s, 1H), 10.92 (s, 1H).

Anal. (C₂₂H₂₄ClN₃O₄S) C, H, N.

In the same manner as in Step 1 except that 4-fluorobenzenesulfonylchloride was used instead of 4-chlorobenzenesulfonyl chloride of Step 1,N-(4-fluorophenylsulfonyl)-L-valyl-L-tryptophanal (Compound 49) wasobtained as white crystals. [Step 2]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.80 (d, 3H, J=6.8 Hz), 0.81 (d, 3H, J=6.8Hz), 1.76-1.88 (m, 1H), 2.82 (dd, 1H, J=8.1, 15.1 Hz), 3.06 (dd, 1H,J=6.1, 15.1 Hz), 3.63 (dd, 1H, J=6.8, 9.3 Hz) 4.04-4.12 (m, 1H),6.98-7.56 (m, 7H), 7.68-7.76 (m, 2H), 7.93 (d, 1H, J=9.3 Hz), 8.41 (d,1H, J=6.4 Hz), 9.19 (s, 1H), 10.92 (s, 1H).

Anal. (C₂₂H₂₄FN₃O₄S) C, H, N.

In the same manner as in Step 1 except that 2-naphthalenesulfonylchloride was used instead of 4-chlorobenzenesulfonyl chloride, and1-aminocyclohexanecarboxylic acid was used instead of valine of Step 1,1-(2-naphthalenesulfonylamino)cyclohexanecarbonyl-L-tryptophanal(Compound 50) was obtained as white crystals. [Step 3]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 1.17 (s br, 6H), 1.72 (s br, 4H), 2.97-3.16(m, 2H), 4.10-4.17 (m, 1H), 6.95-7.22 (m, 3H), 7.33 (d, 1H, J=8.3 Hz),7.48 (d, 1H, J=7.6 Hz), 7.61-7.72 (m, 2H), 7.83-8.14 (m, 6H), 8.41 (s,1H), 10.89 (s, 1H).

Anal. (C₂₈H₂₉N₃O₄S) C, H, N.

In the same manner as in Step 1 except that 2-naphthalenesulfonylchloride was used instead of 4-chlorobenzenesulfonyl chloride, andtert-leucine was used instead of valine of Step 1,N-(2-naphthalene-sulfonyl)-L-tert-leucyl-L-tryptophanal (Compound 51)was obtained as white crystals.

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.89 (s, 9H), 2.43 (dd, 1H, J=6.8, 15.1 Hz),2.68 (dd, 1H, J=7.3, 15.1 Hz), 3.64-3.75 (m, 2H), 6.93-7.16 (m, 3H),7.19 (d, 1H, J=7.8 Hz), 7.32 (d, 1H, J=8.3 Hz), 7,58-7.67 (m, 2H),7.76-7.80 (m, 2H), 7.88-8.01 (m, 3H), 8.05-8.09 (m, 1H), 8.37 (d, 1H,J=6.4 Hz), 8.43 (m, 1H), 8.83 (s, 1H), 10.80 (s, 1H).

Anal. (C₂₇H₂₉N₃O₄S) C, H, N.

Example 54 N-(4-Fluorophenylsulfonyl)-L-valyl-L-cyclohexylalaninal

Valine (11.5 g) was dissolved in 1M aqueous sodium hydroxide solution(100 ml), and purified water (200 ml) and tetrahydrofuran (100 ml) wereadded. Thereto were simultaneously added dropwise 1M aqueous sodiumhydroxide solution (100 ml) and a solution (100 ml) of4-fluorobenzenesulfonyl chloride (17.5 g) in tetrahydrofuran withstirring under ice-cooling. The solution was stirred for one day at roomtemperature to allow reaction. After the completion of the reaction, thereaction mixture was adjusted to pH 2-3 and extracted with ethylacetate. The extract was washed with dilute hydrochloric acid andsaturated brine, and dried over anhydrous magnesium sulfate. Ethylacetate was evaporated under reduced pressure, and the residue waswashed with a mixture of hexane-ethyl acetate to give 15.5 g ofN-(4-fluorophenylsulfonyl)-L-valine as white crystals.

N-(4-Fluorophenylsulfonyl)-L-valine (12.0 g) and N-hydroxysuccinimide(7.6 g) were dissolved in tetrahydrofuran (200 ml), and a solution (200ml) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (12.6g) in dichloromethane was gradually added dropwise with stirring underice-cooling. The solution was stirred at room temperature for about 4 hrto allow reaction. After the completion of the reaction, the solvent wasevaporated under reduced pressure and the residue was dissolved in ethylacetate. The mixture was washed with dilute hydrochloric acid, saturatedaqueous solution of sodium hydrogencarbonate and saturated brine, anddried over anhydrous magnesium sulfate. Ethyl acetate was evaporatedunder reduced pressure, and the residue was washed with a mixed solutionof hexane-ethyl acetate to give 14.1 g ofN-(4-fluorophenylsulfonyl)-L-valine N-hydroxysuccinimide ester as whitecrystals.

N-(4-Fluorophenylsulfonyl)-L-valine N-hydroxysuccinimide ester (1.5 g)and (S)-2-amino-3-cyclohexyl-1-propanol hydrochloride (1.5 g) were addedto dichloromethane (80 ml), and the mixture was stirred at roomtemperature while adding triethylamine (2.0 g). The solution was stirredfor 2 hr to allow reaction. After the completion of the reaction, thesolvent was evaporated under reduced pressure. The residue was dissolvedin ethyl acetate, washed with dilute hydrochloric acid, saturatedaqueous solution of sodium hydrogencarbonate and saturated brine, anddried over anhydrous magnesium sulfate. Ethyl acetate was evaporatedunder reduced pressure, and the residue was washed with a mixed solutionof hexane-ethyl acetate to give 1.4 g ofN-(4-fluorophenylsulfonyl)-L-valyl-L-cyclohexylalaninol as whitecrystals.

N-(4-Fluorophenylsulfonyl)-L-valyl-L-cyclohexylalaninol (1.3 g) wasdissolved in dimethyl sulfoxide (20 ml) and dichloromethane (10 ml), andtriethylamine (1.9 g) was added. The solution was stirred at roomtemperature while adding a solution (10 ml) of sulfur trioxide-pyridinecomplex (2.0 g) in dimethyl sulfoxide, which was followed by stirringfor 1 hr. After the completion of the reaction, ethyl acetate was added.The mixture was washed with dilute hydrochloric acid, saturated aqueoussolution of sodium hydrogencarbonate and saturated brine, and dried overanhydrous magnesium sulfate. The solvent was evaporated under reducedpressure, and the residue was purified by preparative TLC plate(developing solvent: hexane-ethyl acetate, 1:1, v/v) to give 0.37 g ofN-(4-fluorophenylsulfonyl)-L-valyl-L-cyclohexylalaninal (Compound 52) aswhite crystals. [Step 1]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.74-1.61 (m, 13H), 0.82 (d, 3H, J=10.9 Hz),0.84 (d, 3H, J=10.9 Hz), 1.80-1.93 (m, 1H), 3.53-3.66 (m, 1H), 3.77-3.85(m, 1H), 7.32-7.42 (m, 2H), 7.79-7.87 (m, 2H), 7.96 (d, 1H, J=8.9 Hz),8.29 (d, 1H, J=6.6 Hz), 9.10 (s, 1H).

Anal. (C₂₀H₂₉FN₂O₄S) C, H, N.

In the same manner as in Step 1 except that 2-naphthalenesulfonylchloride was used instead of 4-fluorobenzenesulfonyl chloride,N-(2-naphthalenesulfonyl)-L-valyl-L-cyclohexylalaninal (Compound 53) wasobtained as white crystals.

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.52-0.82 (m, 13H), 0.82 (d, 3H, J=6.6 Hz),0.84 (d, 3H, J=5.6 Hz), 1.81-1.99 (m, 1H), 3.63-3.69 (m, 2H), 7.80 (dd,1H, J=1.9, 8.8 Hz), 8.00-8.11(m, 4H), 8.26 (d, 1H, J=6.6 Hz), 8.39 (m,1H), 8.96 (s, 1H).

Anal. (C₂₄H₃₂N₂O₄S) C, H, N.

In the same manner as in Step 1 except that 4-chlorophenylsulfonylchloride was used instead of 4-fluorobenzenesulfonyl chloride,N-(4-chlorophenylsulfonyl)-L-valyl-L-cyclohexylalaninal (Compound 54)was obtained as white crystals.

¹H-NMR DMSO-d₆ 270 MHz) δ: 0.74-1.61 (m, 13H), 0.82 (d, 3H, J=10.2 Hz),0.85 (d, 3H, J=10.5 Hz), 1.89-1.93 (m, 1H), 3.58-3.63 (m, 1H), 3.77-3.85(m, 1H), 7.58-7.63 (m, 2H), 7.75-7.80 (m, 2H), 8.05 (d, 1H, J=7.3 Hz),8.40 (d, 1H, J=6.6 Hz), 9.11 (s, 1H).

Anal. (C₂₀H₂₉Cl-N₂O₄S) C, H, N.

Example 55 N-(4-Fluorophenylsulfonyl)-D-valyl-D-leucinal

D-Valine (6.6 g) was dissolved in 1M aqueous sodium hydroxide solution(50 ml), and purified water (200 ml) and tetrahydrofuran (100 ml) wereadded. Thereto were simultaneously added dropwise 1M aqueous sodiumhydroxide solution (100 ml) and a solution (50 ml) of4-fluorobenzenesulfonyl chloride (9.7 g) in tetrahydrofuran withstirring under ice-cooling. The solution was stirred for one day at roomtemperature to allow reaction. After the completion of the reaction, thereaction mixture was adjusted to pH 2-3 and extracted with ethylacetate. The extract was washed with dilute hydrochloric acid andsaturated brine, and dried over anhydrous magnesium sulfate. Ethylacetate was evaporated under reduced pressure, and the residue waswashed with a mixture of hexane-ethyl acetate to give 8.3 g ofN-(4-fluorophenylsulfonyl)-L-valine as white crystals.

N-(4-Fluorophenylsulfonyl)-L-valine (8.0 g) and N-hydroxysuccinimide(4.4 g) were dissolved in tetrahydrofuran (150 ml), and a solution (150ml) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (7.3g) in dichloromethane was gradually added with stirring underice-cooling. The solution was stirred at room temperature for about 12hr to allow reaction. After the completion of the reaction, the solventwas evaporated under reduced pressure and the residue was dissolved inethyl acetate. The mixture was washed with dilute hydrochloric acid,saturated aqueous solution of sodium hydrogencarbonate and saturatedbrine, and dried over anhydrous magnesium sulfate. Ethyl acetate wasevaporated under reduced pressure, and the residue was washed with amixed solution of hexane-ethyl acetate to give 9.6 g ofN-(4-fluorophenylsulfonyl)-D-valine N-hydroxysuccinimide ester as whitecrystals.

N-(4-Fluorophenylsulfonyl)-D-valine N-hydroxysuccinimide ester (1.8 g)and D-leucinol (0.74 g) were added to dichloromethane (80 ml), and themixture was stirred at room temperature while adding triethylamine (1.5g). The solution was stirred for 2 hr to allow reaction. After thecompletion of the reaction, the solvent was evaporated under reducedpressure. The residue was dissolved in ethyl acetate, washed with dilutehydrochloric acid, saturated aqueous solution of sodiumhydrogencarbonate and saturated brine, and dried over anhydrousmagnesium sulfate. Ethyl acetate was evaporated under reduced pressure,and the residue was washed with a mixed solution of hexane-ethyl acetateto give 1.6 g of N-(4-fluorophenylsulfonyl)-D-valyl-D-leucinol as whitecrystals.

N-(4-Fluorophenylsulfonyl)-D-valyl-D-leucinol (1.5 g) was dissolved indimethyl sulfoxide (20 ml) and dichloromethane (10 ml), andtriethylamine (2.4 g) was added. The solution was stirred at roomtemperature while adding a solution (20 ml) of sulfur trioxide-pyridinecomplex (2.6 g) in dimethyl sulfoxide, which was followed by stirringfor 1 hr. After the completion of the reaction, ethyl acetate was added.The mixture was washed with dilute hydrochloric acid, saturated aqueoussolution of sodium hydrogencarbonate and saturated brine, and dried overanhydrous magnesium sulfate. The solvent was evaporated under reducedpressure, and the residue was purified by preparative TLC plate(developing solvent: hexane-ethyl acetate, 1:1, v/v) to give 1.0 g ofN-(4-fluorophenylsulfonyl)-D-valyl-D-leucinal (Compound 55) as whitecrystals. [Step 1]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.74 (d, 3H, J=6.3 Hz), 0.82 (d, 6H, J=6.3Hz), 0.87 (d, 3H, J=6.9 Hz), 1.15-1.45 (m, 3H), 1.81-1.93 (m, 1H), 3.59(t, 1H, J=6.8 Hz), 3.80-3.88 (m, 1H), 7.33-7.42 (m, 2H), 7.79-7.86 (m,2H), 7.95 (d, 1H, J=6.9 Hz), 8.26 (d, 1H, J=6.9 Hz), 9.14 (s, 1H).

Anal. (C₂₂H₃₀N₂O₄S) C, H, N.

In the same manner as in Step 1 except that valine was used instead ofD-valine, N-(4-fluorophenylsulfonyl)-L-valyl-D-leucinal (Compound 56)was obtained as white crystals. [Step 2]

¹H-NMR (DMSO-d₆ 270 MHz) δ: 0.78 (d, 3H, J=6.3 Hz), 0.82 (d, 3H, J=6.6Hz), 0.83 (d, 6H, J=6.3 Hz), 1.18-1.50 (m, 3H), 1.79-1.92 (m, 1H),3.61-3.63 (m, 1H), 3.84-3.92 (m, 1H), 7.33-7.44 (m, 2H), 7.80-7.96 (m,3H), 8.22 (d, 1H, J=6.9 Hz), 8.96 (s, 1H).

Anal. (C₂₀H₂₉FN₂O₄S) C, H, N.

Example 56 (Tablet)

Compound 35 30 mg Lactose 80 mg Starch 17 mg Magnesium stearate 3 mg

Using the above ingredient as the material for one tablet, tablets areprepared by a conventional method. Where necessary, sugar coating may beapplied.

Example 57 (Injection)

Compound 48 2.5 mg Sodium chloride 900 mg 1N sodium hydroxide q.s.Distilled water for injection total 100 ml

The above ingredients are admixed by a conventional method to giveinjections.

Example 58 (Eye drop)

Compound 35 50 mg Boric acid 700 mg Sodium tetraborate q.s. Sodiumchloride 500 mg Hydroxymethylcellulose 500 mg Disodium edetate 0.05 mgBenzalkonium chloride 0.005 mg Sterile purified water total 100 ml

The above ingredients are admixed by a conventional method to give asuspension for eye drop.

Experimental Example 1

Effect of cysteine protease inhibitor on angiogenesis in cornea ofguinea pig by transplantation of basic FGF (bFGF)

(Test Method)

(1) Method for preparing pellets for transplantation

A solution (5 μl) of 8% ethylene vinyl acetate copolymer (EVA) indichloromethane was dropped on a plate made of Tefron, air-dried, and0.0167 w/v % bFGF (3 μl) was dropped thereon and air-dried. Afterdrying, the obtained product was rounded into a small rod to give a bFGF(500 ng)-containing pellet.

Using a test solution instead of 0.0167 w/v % bFGF, a testsolution-containing pellet was prepared in the same manner as above. Thetest solution-containing pellets were prepared to contain 27 mercalpastatin peptide (0.03 μmole and 0.1 μmole/pellet, Sigma) andleupeptin (0.1 μmole/pellet, PEPTIDE INSTITUTE, INC.) .

As a control, a pellet made of EVA alone was prepared and used as avehicle pellet.

(2) Transplantation of pellet into cornea of guinea pig

The procedure followed the method of M. Kusaka et al. [Biochem. Biophys.Res. Comm., vol. 174, pp. 1070-1076 (1991)]. That is, male guinea pigsweighing 300-400 g were anesthetized with a 1:1 mixture of Ketalar 50(ketamin hydrochloride, Sankyo Company, Limited) and Celactal (xylazinehydrochloride, Bayer, Ltd.). A pocket was formed in the intercellularlayer of corneal stroma layer of both eyes from corneal limbus to thecenter of cornea, using a 0.5 mm width ophthalmic spatula. A testsolution-containing pellet was inserted into the pocked thus formed, anda bFGF-containing pellet was inserted in adjacency thereto. Forprevention of infection, ofloxacin eye ointment [Tarivid eye ointment(ofloxacin 0.3%, manufactured by Santen Pharmaceutical Co., Ltd.) wasinstilled once immediately after insertion of the pellets. Thereafter,0.3% lomefloxacin (lomeflon ophthalmic otologic solution, manufacturedby Senju Pharmaceutical Co., Ltd.) was instilled once a day for 5 days.

(3) Evaluation of the effects of cysteine protease inhibitor

The effect of the cysteine protease inhibitor was observed with a slitlamp. The blood vessel newly formed in cornea which is an avasculartissue had high permeability, and the wet weight and plasma content wereexpected to elevate due to incurrent of plasma components as a result ofhigh permeability. At 9 days posttransplantation of pellet, guinea pigswere euthanized and cornea was collected. The obtained cornea wasweighed (wet weight). Then, it was homogenized, and subjected tocentrifugation. The obtained water soluble protein was separated by SDSpolyacrylamide gel electrophoresis. After electrophoresis, the gel wasstained with Coomassie Brilliant Blue. By image analysis (NIH Image1.31), albumin which is one of the major proteins in plasma wasquantitatively assayed. As the standard substance, guinea pig serumalbumin was used.

(Test Results)

(1) Observation of cornea with a slit lamp

The cornea of vehicle pellet transplantation group showed no changes ascompared to the cornea of the untreated group. The bFGF-containingpellet transplantation group (hereinafter sometimes referred to ascontrol group) showed appearance of blood vessel in the neighborhood ofbFGF-containing pellet transplantation site from 4 days aftertransplantation. At 9 days posttransplantation, blood vessels were newlyformed radially in the entirety of the cornea of guinea pigs. The groupwhich underwent transplantation of 27 mer calpastatin peptide-containingpellet or leupeptin-containing pellet together with bFGF-containingpellet showed appearance of blood vessel in all groups. Compared to thecontrol group, however, the degree of appearance was mild, and newformation of blood vessels was found mainly in corneal limbus. Thecorneas at 9 days posttransplantation of guinea pig are shown in FIGS. 1and 2.

(2) Wet weight of cornea

The results are shown in FIG. 3. The wet weight of cornea was almost thesame between the untreated group and vehicle pellet transplantationgroup, and no influence by transplantation of vehicle pellet was found.In contrast, the wet weight of cornea of the control group increasedfrom the weight of the untreated group and vehicle pellettransplantation group. The group which underwent transplantation of 27mer calpastatin peptide-containing pellet or leupeptin-containing pellettogether with bFGF-containing pellet showed suppressed increase in thewet weight of cornea as compared to the control group.

It was clarified therefrom that 27 mer calpastatin peptide and leupeptinsuppressed increase in wet weight of cornea caused by angiogenesis.

(3) Amount of albumin in cornea

The results are shown in FIG. 4. The amount of albumin of untreatedgroup was very small and was 35.2±9.6 (S.D.) or 56.1±13.5 (S.D.). Thiswas because cornea is an avascular tissue. Meanwhile, that of the groupwhich underwent transplantation of vehicle pellet showed a smallincrease from the weight of the untreated group. This was supposedlyattributable to surgical stimulation during pellet transplantation. Incontrast, that of the control group was about 17 or 9 times greater thanthe untreated group, and about 8 times greater than the vehicle pellettransplantation group. This was because vascular permeability was highin newly formed blood vessels, and albumin which is the main componentof plasma leaked. The group transplanted with 27 mer calpastatinpeptide-containing pellet and leupeptin-containing pellet together withbFGF-containing pellet showed suppressed increase in the amount ofalbumin in cornea as compared to the control group.

It was clarified from the above that a cysteine protease inhibitorsuppressed angiogenesis.

Experimental Example 2

The biological activity of the compounds of the formula (I) and (IV) isshown in the following. The compounds of the formula (I) and (IV) andsalts thereof show thiol protease-inhibitory activity. The inhibitoryactivity against calpain, cathepsin L, papain, and trypsin which is aserine protease was determined. The results are shown in Tables 6 and 7.

μ-Calpain-inhibitory activity

The activity of μ-calpain (Nakalai Tesque) was assayed in accordancewith the procedure described in the literature [Anal. Biochem., 208,387-392 (1993)]. Thus, to a solution containing 0.5 mg/ml casein, 50 mMTris-HCl (pH 7.4), 20 mM dithiothreitol, and 4 mM calcium chloride wasadded 2.5 μl of a dimethyl sulfoxide solution containing a varyingconcentration of the test drug as well as 0.03 unit of μ-calpain toinitiate the reaction. The final liquid volume was 250 μl. After 60minutes of reaction at 30° C., 100 μl of the reaction mixture wastransferred to another vessel, to which 50 μl of purified water and 100μl of 50% Coumassie brilliant blue solution were added. The mixture wasallowed to stand at room temperature for 15 minutes and the absorbancewas measured at 595 nm. As a control, 2.5 μl of dimethyl sulfoxide notcontaining the test drug was added and the mixture was treated in thesame manner as above. The absorbance value thus found was used as thecontrol value. Similarly, the value found by adding 0.2 mM EDTA in lieuof 4 mM aqueous calcium chloride solution was used as the blank value.The inhibition rate was calculated by means of the following equationand plotted against concentration on log paper and the amount necessaryfor 50% inhibition (IC₅₀) was determined.${{Inhibition}\quad {rate}\quad (\%)} = {\left( {1 - \frac{{{Measured}\quad {value}} - {{blank}\quad {value}}}{{{Control}\quad {value}} - {{blank}\quad {value}}}} \right) \times 100}$

Assay of cathepsin L-inhibitory activity

The activity of cathepsin L (Cosmo Bio), a cysteine protease, wasassayed by the method described in the literature [Methods inEnzymology, 80, 535-561, 1981]. Thus, to a solution containing 85 mMacetate buffer (pH 5.5), 2 mM dithiothreitol, 1 mM EDTA, 2 μg cathepsinL, and a varying concentration of the test compound was added 50 μl of20 μM carbobenzoxy-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide(Z-Phe-Arg-MCA) to initiate the reaction at the final liquid volume of200 μl. After 20 minutes of reaction at 30° C., 20 μl of 1 M Tris-HCl(pH 8.0) was added so as to stop the reaction. The amount of liberated4-methyl-7-aminocoumarin was determined with a fluorospectrometer at anexcitation wavelength of 360 nm and a fluorescent emission wave lengthof 450 nm. Using the value found without addition of the test drug ascontrol and the value found without addition of the enzyme as blank,IC₅₀ was determined in the same manner as above.

Assay of papain- and trypsin-inhibitory activity

The activity of papain which is a cysteine protease and of trypsin(Sigma) which is a serine protease was assayed in accordance with themethod described in the literature [Anal. Biochem., 208, 387-392, 1993].Thus, to a solution containing 0.5 mg/ml casein, 50 mM Tris-HCl (pH8.0), 20 mM dithiothreitol, and 0.2 mM EDTA was added 2.5 μl of dimethylsulfoxide containing a varying concentration of the test drug as well as0.03 unit of papain or trypsin to initiate the reaction. The finalliquid volume was adjusted to 250 μl. After 60 minutes of reaction at30° C., 100 μl of the reaction mixture was transferred to another vesseland following addition of 50 μl of purified water and 100 μl of 50%Coumassie brilliant blue solution, the mixture was allowed to stand atroom temperature for 15 minutes. The absorbance of the mixture was thenmeasured at 595 nm. Using the value found similarly by adding 2.5 μl ofdimethyl sulfoxide not containing the test drug as control and the valuefound without addition of the enzyme as blank, IC50 was determined inthe same manner as above.

TABLE 6 Calpain 50% inhibitory concentration (IC₅₀) Test drug (M) Testdrug (M) Compound  3 4.7 × 10⁻⁵ Compound 34 1.0 × 10⁻⁸ Compound 11 2.9 ×10⁻⁶ Compound 35 7.5 × 10⁻⁹ Compound 13 8.l × 10⁻⁷ Compound 36 3.1 ×10⁻⁸ Compound 14 7.8 × 10⁻⁷ Compound 37 2.8 × 10⁻⁸ Compound 15 1.1 ×10⁻⁶ Compound 38 6.1 × 10⁻⁶ Compound 16 6.4 × 10⁻⁷ Compound 39 4.2 ×10⁻⁶ Compound 17 3.5 × 10⁻⁷ Compound 40 2.6 × 10⁻⁷ Compound 18 6.3 ×10⁻⁷ Compound 41 1.3 × 10⁻⁷ Compound 19 4.9 × 10⁻⁷ Compound 42 3.2 ×10⁻⁶ Compound 20 1.2 × 10⁻⁶ Compound 43 2.7 × 10⁻⁸ Compound 21 9.5 ×10⁻⁷ Compound 44 1.4 × 10⁻⁸ Compound 22 1.8 × 10⁻⁵ Compound 45 1.4 ×10⁻⁸ Compound 23 5.1 × 10⁻⁶ Compound 46 1.8 × 10⁻⁸ Compound 24 8.4 ×10⁻⁷ Conpound 47 2.0 × 10⁻⁷ Compound 25 4.1 × 10⁻⁵ Compound 48 1.3 ×10⁻⁸ Compound 27 2.4 × 10⁻³ Compound 49 2.3 × 10⁻⁸ Compound 28 3.9 ×10⁻⁶ Compound 50 3.6 × 10⁻⁷ Compound 29 6.0 × 10⁻⁷ Compound 51 1.1 ×10⁻⁶ Compound 30 6.0 × 10⁻⁶ Compound 52 3.0 × 10⁻⁸ Compound 31 1.5 ×10⁻⁴ Compound 53 8.3 × 10⁻⁹ Compound 33 1.9 × 10⁻⁶ Compound 54 1.4 ×10⁻⁸ Compound 55 1.0 × 10⁻⁴ Compound 56 1.4 × 10⁻⁶

TABLE 7 50% Inhibitory concentration (IC₅₀) Test drug Cathepsin L PapainTrypsin (Compound No.) (M) (M) (M) Compound 11 1.2 × 10⁻⁸ 1.1 ×10⁻⁷ >3.0 × 10⁻³ Compound 13 2.9 × 10⁻⁸ 7.9 × 10⁻⁸ >3.0 × 10⁻³ Compound16 8.2 × 10⁻⁸ 2.1 × 10⁻⁷ >3.0 × 10⁻³ Compound 17 2.7 × 10⁻⁸ 6.2 ×10⁻⁸ >3.0 × 10⁻³ Compound 22 3.8 × 10⁻⁵ 4.0 × 10⁻⁵ >3.0 × 10⁻³ Compound24 9.0 × 10⁻⁹ 4.7 × 10⁻⁸ >3.0 × 10⁻³

As is evident from the above experimental results, the cysteine proteaseinhibitory compound to be used in the present invention showed notoxicity to human and animals.

Having inhibitory activity against cysteine proteases such as calpain,cathepsin L and papain and showing no activity against serine protease(trypsin), the compounds of formulas (I) and (IV) and salts thereof areuseful as prophylactic or therapeutic agents for a variety of cysteineprotease-associated diseases, such as ischemic diseases, inflammatorydiseases, muscular dystrophy, cataract, immune diseases, essentialhypertension, Alzheimer's disease, subarachnoid hemorrhage, andosteoporosis, in mammals (e.g. mouse, rat, rabbit, dog, cat, bovine,swine, and human).

The angiogenesis inhibitor of the present invention suppresses newformation of blood vessels in the living tissues, so that it can be usedas a superior therapeutic or prophylactic agent of angiogenesisassociated with wound healing, inflammation, growth of tumor and thelike; and angiogenesis as seen in diabetic retinopathy, prematurityretinopathy, retinal venous occlusion, senile discoid maculardegeneration and the like, as well as for prevention of metastasis oftumors.

8 12 amino acids amino acid single linear peptide unknown 1 Gly Lys ArgGlu Val Thr Ile Pro Pro Lys Tyr Arg 1 5 10 12 amino acids amino acidsingle linear peptide unknown 2 Gly Lys Arg Glu Val Thr Leu Pro Pro LysTyr Arg 1 5 10 12 amino acids amino acid single linear peptide unknown 3Gly Glu Asp Asp Glu Thr Ile Pro Ser Glu Tyr Arg 1 5 10 12 amino acidsamino acid single linear peptide unknown 4 Gly Glu Asp Asp Glu Thr ValPro Pro Glu Tyr Arg 1 5 10 12 amino acids amino acid single linearpeptide unknown 5 Gly Glu Asp Asp Glu Thr Val Pro Ala Glu Tyr Arg 1 5 1012 amino acids amino acid single linear peptide unknown 6 Gly Glu LysGlu Glu Thr Ile Pro Pro Asp Tyr Arg 1 5 10 12 amino acids amino acidsingle linear peptide unknown 7 Gly Glu Arg Asp Asp Thr Ile Pro Pro GluTyr Arg 1 5 10 27 amino acids amino acid single linear peptide unknown 8Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly Lys Arg Glu 1 5 10 15Val Thr Ile Pro Pro Lys Tyr Arg Glu Leu Leu Ala 20 25

What is claimed is:
 1. A compound of the formula (VI):

wherein R¹¹ is an optionally substituted aryl having 6 to 10 carbon atoms; R¹² and R¹³ are the same or different and each is a hydrogen, a C₁-C₄ alkyl, or R¹² and R¹³ combinedly form a ring having 3 to 7 carbon atoms; and R¹⁴ is a lower alkyl optionally substituted by aryl, cycloalkyl or aromatic heterocycle, or a salt thereof.
 2. The compound of claim 1, wherein R¹¹ is phenyl or naphthyl optionally substituted by fluorine, chlorine or methyl, or a salt thereof.
 3. The compound of claim 2, wherein R¹¹ is a member selected from 4-fluorophenyl, 4-chlorophenyl, p-tolyl and 2-naphthyl, or a salt thereof.
 4. The compound of claim 1, wherein R¹² is propyl, isopropyl or tert-butyl, and R¹³ is hydrogen, or a salt thereof.
 5. The compound of claim 4, wherein R¹² is isopropyl and R¹³ is hydrogen, or a salt thereof.
 6. The compound of claim 1, wherein R¹² and R¹³ combinedly form cyclohexylidene, or a salt thereof.
 7. The compound of claim 1, wherein R¹⁴ is isobutyl, benzyl, cyclohexylmethyl or indol-3-ylmethyl, or a salt thereof.
 8. A pharmaceutical composition comprising the compound according to claim 1 together with a pharmaceutical carrier.
 9. A pharmaceutical composition comprising the compound according to claim 2 together with a pharmaceutical carrier.
 10. A pharmaceutical composition comprising the compound according to claim 3 together with a pharmaceutical carrier.
 11. A pharmaceutical composition comprising the compound according to claim 4 together with a pharmaceutical carrier.
 12. A pharmaceutical composition comprising the compound according to claim 5 together with a pharmaceutical carrier.
 13. A pharmaceutical composition comprising the compound according to claim 6 together with a pharmaceutical carrier.
 14. A pharmaceutical composition comprising the compound according to claim 7 together with a pharmaceutical carrier.
 15. A pharmaceutical composition comprising the compound according to claim 1 together with at least one vehicle, lubricant, disintegrator, binder, stabilizer, suspending agent, corrigent, aromatic, solvent, isotonizing agent, pH adjusting agent, suppository base, ointment base, wetting agent, emulsifier, surfactant, preservative or buffer.
 16. A pharmaceutical composition comprising the compound according to claim 2 together with at least one vehicle, lubricant, disintegrator, binder, stabilizer, suspending agent, corrigent, aromatic, solvent, isotonizing agent, pH adjusting agent, suppository base, ointment base, wetting agent, emulsifier, surfactant, preservative or buffer.
 17. A pharmaceutical composition comprising the compound according to claim 3 together with at least one vehicle lubricant, disintegrator, binder, stabilizer, suspending agent, corrigent, aromatic, solvent, isotonizing agent, pH adjusting agent, suppository base, ointment base, wetting agent, emulsifier, surfactant, preservative or buffer.
 18. A pharmaceutical composition comprising the compound according to claim 4 together with at least one vehicle, lubricant, disintegrator, binder, stabilizer, suspending agent, corrigent, aromatic, solvent, isotonizing agent, pH adjusting agent, suppository base, ointment base, wetting agent, emulsifier, surfactant, preservative or buffer.
 19. A pharmaceutical composition comprising the compound according to claim 5 together with at least one vehicle, lubricant, disintegrator, binder, stabilizer, suspending agent, corrigent, aromatic, solvent, isotonizing agent, pH adjusting agent, suppository base, ointment base, wetting agent, emulsifier, surfactant, preservative or buffer.
 20. A pharmaceutical composition comprising the compound according to claim 6 together with at least one vehicle, lubricant, disintegrator, binder, stabilizer, suspending agent, corrigent, aromatic, solvent, isotonizing agent, pH adjusting agent, suppository base, ointment base, wetting agent, emulsifier, surfactant, preservative or buffer.
 21. A pharmaceutical composition comprising the compound according to claim 7 together with at least one vehicle, lubricant, disintegrator, binder, stabilizer, suspending agent, corrigent, aromatic, solvent, isotonizing agent, pH adjusting agent, suppository base, ointment base, wetting agent, emulsifier, surfactant, preservative or buffer. 